TW202421656A - Combination therapies for the treatment of cancer - Google Patents

Abstract

Provided are methods of treating cancer ( e.g., a cancer that comprises one or more cancer cells that express a KRAS G12C mutant protein and/or have at least one mTOR-activating aberration) in an individual that comprise administering a composition comprising nanoparticles that comprise an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative thereof) and an albumin in combination with a KRAS G12C inhibitor to the individual. Also provided are related kits.

Description

Combination therapy for cancer treatment

This application relates to combination therapy for the treatment of cancer, such as cancer characterized by expression of KRAS mutant protein (e.g., KRAS G12C mutant protein) and dysregulated (e.g., activated) mTOR signaling.

Mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that acts as a signaling hub in cells to integrate intracellular and extracellular signals and regulate cell growth and homeostasis. Activation of the mTOR pathway is associated with cell proliferation and survival, while inhibition of mTOR signaling causes inflammation and cell death. Dysregulation of the mTOR signaling pathway is associated with an increasing number of human diseases, including cancer and autoimmune disorders. Therefore, mTOR inhibitors have found wide application in the treatment of different pathological conditions, such as solid tumors, hematological malignancies, organ transplantation, restenosis, and rheumatoid arthritis.

Sirolimus (INN/USAN), also known as rapamycin, is an immunosuppressive drug used to prevent organ transplant rejection; it is particularly useful in kidney transplants. Sirolimus dissolving stent is approved in the United States for the treatment of coronary artery restenosis. In addition, sirolimus has been shown to be an effective inhibitor of tumor growth in various cell lines and animal models. Other limus drugs, such as sirolimus analogs, have been designed to improve the pharmacokinetic and pharmacodynamic properties of sirolimus. For example, temsirolimus is approved in the United States and Europe for the treatment of renal cell carcinoma. Everolimus is approved in the United States for the treatment of advanced breast cancer, pancreatic neuroendocrine tumors, advanced renal cell carcinoma, and subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis. The mode of action of sirolimus is binding to the cytoplasmic protein FK binding protein 12 (FKBP12), and the sirolimus-FKBP12 complex then inhibits the mTOR pathway by directly binding to mTOR complex 1 (mTORC1).

Albumin-based nanoparticle compositions have been developed as drug delivery systems for the delivery of substantially water-insoluble drugs. See, e.g., U.S. Patent Nos. 5,916,596; 6,506,405; 6,749,868, and 6,537,579, 7,820,788, and 7,923,536. Abraxane® is an albumin-stabilized nanoparticle composition containing paclitaxel that was approved in the U.S. in 2005 and subsequently in several other countries for the treatment of metastatic breast cancer, pancreatic cancer, and non-small cell lung cancer. FYARRO® is an albumin-stabilized nanoparticle composition containing sirolimus, recently approved for the treatment of perivascular epithelioid cell neoplasm (PEcoma).

KRAS mutations also play a role in some of the most common and deadly cancers, including lung, colorectal, and colorectal cancers. KRAS mutations are estimated to be present in approximately 25% of tumors. A single type of KRAS mutation, the KRAS G12C mutation, accounts for approximately 44% of all KRAS mutations. G12C is a single-point mutation that substitutes a glycine to a cysteine at codon 12 of the KRAS protein. This substitution favors the active GTP-bound conformation of KRAS, thereby amplifying the signaling pathway that leads to tumorigenesis. KRAS G12C is particularly prevalent in non-small cell lung cancer (NSCLC), which accounts for approximately 85% of all lung cancer cases in the United States. Approximately 13% of Americans with NSCLC have the KRAS G12C mutation, and approximately 23,000 new cases of KRAS G12C NSCLC are diagnosed each year in the U.S. alone. Clinical trials of the KRAS G12C allele-specific inhibitors adagrasib and sotorasib have demonstrated promising activity in cancers expressing the KRAS G12C mutant protein. However, clinical trial data also suggest that there is significant variability in responses among patients treated with KRAS G12C inhibitors and that KRAS G12C inhibitor monotherapy is unlikely to be sufficient to induce sustained treatment responses.

Therefore, there remains a need in the art for methods of treating cancers that exhibit dysregulation of the mTOR signaling pathway and express the KRAS G12C mutant protein.

All references cited herein (including patent applications, patent publications, and UniProtKB/Swiss-Prot accession numbers) are hereby incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual: (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin and (b) an effective amount of a KRAS inhibitor. In some embodiments, the cancer comprises one or more cancer cells expressing a KRAS mutant protein. In some embodiments, the cancer comprises (e.g., further comprises) one or more cancer cells having at least one mTOR activating aberration. In some embodiments, the individual is a human.

In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the average diameter of the nanoparticles in the composition does not exceed about 150 nm. In some embodiments, the average diameter of the nanoparticles in the composition does not exceed about 120 nm. In some embodiments, the weight ratio of albumin to mTOR inhibitor in the nanoparticle composition does not exceed about 10:1. In some embodiments, the nanoparticles include an mTOR inhibitor conjugated to albumin. In some embodiments, the nanoparticles include an mTOR inhibitor coated with albumin. In some embodiments, the mTOR inhibitor nanoparticle composition is administered intravenously or subcutaneously. In some embodiments, the mTOR inhibitor nanoparticle composition is administered intravenously.

In some embodiments, the KRAS inhibitor is an antibody, peptide, protein, antisense oligonucleotide or small molecule that inhibits the activity of KRAS mutant protein. In some embodiments, the KRAS inhibitor is a small molecule. In some embodiments, the KRAS inhibitor is a small molecule KRAS G12C inhibitor selected from the group consisting of: sotolacib, adagracib, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, RMC-8839, HBI-2438 and JNJ-74699157. In some embodiments, the small molecule KRAS G12C inhibitor is sotolacib or adagracib. In some embodiments, sotolacib or adagracib is administered orally. In some embodiments, the cancer comprises one or more cancer cells expressing KRAS G12C mutant proteins. In some embodiments, the KRAS inhibitor is a small molecule KRAS G12D inhibitor selected from the group consisting of: MRTX1133 and RMC-6236. In some embodiments, the cancer comprises one or more cancer cells expressing KRAS G12D mutant proteins. In some embodiments, the KRAS inhibitor is a small molecule KRAS G12V inhibitor, and wherein the small molecule KRAS G12V inhibitor is JAB-23000. In some embodiments, the cancer comprises one or more cancer cells expressing KRAS G12V mutant proteins.

In some embodiments, the cancer comprising one or more cancer cells expressing KRAS mutant protein and/or having at least one mTOR activation aberration is a solid tumor, lung cancer, bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, uterine cancer, endometrial cancer, cervical cancer, testicular cancer, bile duct cancer, myelodysplastic cancer, or a tumor of unknown origin. In some embodiments, the cancer is a solid tumor, lung cancer, bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, or a tumor of unknown origin. In some embodiments, the cancer or tumor (such as any of the aforementioned cancers or tumors) is advanced, unresectable, and/or metastatic. In some embodiments, the cancer is a solid tumor (e.g., an advanced, unresectable and/or metastatic solid tumor), lung cancer (e.g., advanced, unresectable and/or metastatic lung cancer), or bladder cancer (e.g., advanced, unresectable and/or metastatic bladder cancer). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), e.g., advanced, unresectable and/or metastatic NSCLC.

In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS inhibitor are administered simultaneously. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS inhibitor are administered in parallel. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS inhibitor are administered sequentially. In some embodiments, the mTOR inhibitor nanoparticle composition is administered weekly, once every three weeks, or twice every three weeks. In some embodiments, the KRAS inhibitor is administered daily or twice daily.

In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells having at least one mTOR activating aberration prior to administering the mTOR inhibitor nanoparticle composition and the KRAS inhibitor. In some embodiments, the mTOR activating aberration comprises a mutation in an mTOR-related gene. In some embodiments, the mTOR activating aberration is in at least one mTOR-related gene selected from the group consisting of: AKT1, FLT-3, MTOR, PIK3CA, PIK3CG, TSC1, TSC2, RHEB, STK11, NF1, NF2, TP53, FGFR4, BAP1, KRAS, NRAS, NRF2, KEAP1, and PTEN. In some embodiments, the mTOR activating aberration is in TSC1 and/or TSC2. In some embodiments, the method comprises (e.g., further comprises) selecting an individual for treatment based on the presence of one or more cancer cells expressing a KRAS mutant protein. In some embodiments, the KRAS mutant protein is a KRAS G12C mutant protein, a KRAS G12D mutant protein, or a KRAS G12V mutant protein.

In some embodiments, a kit for treating cancer in an individual is provided, comprising: (a) a composition comprising nanoparticles comprising an mTOR inhibitor and albumin, and (b) instructions for administering an effective amount of the mTOR inhibitor nanoparticle composition and an effective amount of the KRAS inhibitor to an individual having a cancer comprising one or more cancer cells that express KRAS mutant proteins and/or have at least one mTOR activating aberration. In some embodiments, the cancer is a solid tumor, lung cancer, bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, uterine cancer, endometrial cancer, cervical cancer, testicular cancer, bile duct cancer, myelodysplastic cancer, or a tumor of unknown origin. In some embodiments, the individual is a human.

In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the limus drug is sirolimus. In some embodiments, the average diameter of the nanoparticles in the composition does not exceed about 150 nm. In some embodiments, the average diameter of the nanoparticles in the composition does not exceed about 120 nm. In some embodiments, the weight ratio of albumin to mTOR inhibitor in the nanoparticle composition does not exceed about 10:1. In some embodiments, the nanoparticles include an mTOR inhibitor conjugated to albumin. In some embodiments, the nanoparticles include an mTOR inhibitor coated with albumin.

In some embodiments, the KRAS inhibitor is an antibody, peptide, protein, antisense oligonucleotide or small molecule that inhibits the activity of KRAS mutant protein. In some embodiments, the KRAS inhibitor is a small molecule. In some embodiments, the KRAS inhibitor is a small molecule KRAS G12C inhibitor selected from the group consisting of: sotolacib, adagracib, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, RMC-8839, HBI-2438 and JNJ-74699157. In some embodiments, the KRAS inhibitor is a small molecule KRAS G12D inhibitor selected from the group consisting of: MRTX1133 and RMC-6236. In some embodiments, the KRAS inhibitor is a small molecule KRAS G12V inhibitor, and wherein the small molecule KRAS G12V inhibitor is JAB-23000.

Cross-references to related applications

This application claims the benefit of priority to U.S. Patent Application Serial No. 63/415,252 filed on October 11, 2022 and U.S. Patent Application Serial No. 63/461,145 filed on April 21, 2023, the entire contents of which are incorporated herein by reference for all purposes. Overview

This application is based on the unexpected discovery that combination therapy comprising a composition comprising nanoparticles (comprising an mTOR inhibitor and albumin) (e.g., an "mTOR inhibitor nanoparticle composition", such as a sirolimus/albumin nanoparticle composition) and a KRAS inhibitor (e.g., a KRAS G12C inhibitor, such as sotolacizumab or adagracib) is significantly more effective in inhibiting tumor growth than either agent alone. The applicant also found that such combination therapy is more effective in inhibiting tumor growth than combination therapy comprising a non-nanoparticle mTOR inhibitor (e.g., everolimus) and a KRAS inhibitor (e.g., a KRAS G12C inhibitor, such as sotolacizumab or adagracib). Improved tumor growth inhibition (TGI) of combination therapy comprising an mTOR inhibitor nanoparticle composition and a KRAS inhibitor (e.g., a KRAS G12C inhibitor) is associated with significantly higher tumor response rates (e.g., tumor regression with a tumor volume change greater than -30%) than single-agent treatment with an mTOR inhibitor nanoparticle composition, single-agent treatment with a KRAS inhibitor (e.g., a KRAS G12C inhibitor), and combination therapy comprising a non-nanoparticle mTOR inhibitor and a KRAS G12C inhibitor (e.g., a KRAS G12C inhibitor).

Thus, in one aspect, the present application provides a method for treating cancer in an individual, comprising administering to the individual (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin (e.g., nanoparticle albumin-bound sirolimus) and (b) an effective amount of a KRAS inhibitor (e.g., a KRAS G12C inhibitor). In some embodiments, the cancer comprises one or more cells expressing a KRAS mutant protein (e.g., a KRAS G12C mutant protein). The mTOR pathway is often activated in cancer patients with KRAS mutations and contributes to adaptive resistance to KRAS inhibitors (Byun et al. (2019), "Oncogenic KRAS signaling activates mTORC1 via COUP-TFII-mediated lactate production", European Society for Molecular Biology Report 20 (6)). Additionally or alternatively, in some embodiments, the tumor comprises one or more cancer cells having at least one mTOR activating aberration. In another aspect, kits and articles for treating cancer, such as cancers comprising one or more cells expressing KRAS mutant proteins (e.g., KRAS G12C mutant proteins) and/or having at least one mTOR activating aberration, are provided, comprising a composition comprising nanoparticles (comprising an mTOR inhibitor and albumin) (e.g., nanoparticle albumin-bound sirolimus). In some embodiments, kits and articles of manufacture include instructions for administering a composition comprising an mTOR inhibitor and albumin in combination with a KRAS inhibitor (e.g., a KRAS G12C inhibitor) to a subject to treat cancer, such as a cancer comprising one or more cells expressing a KRAS mutant protein (e.g., a KRAS G12C mutant protein) and/or having at least one mTOR activating aberration. In some embodiments, kits and articles of manufacture further comprise a KRAS inhibitor (e.g., a KRAS G12C inhibitor). Definitions

Before describing the embodiments in detail, it should be understood that the present invention is not limited to specific compositions or biological systems, which may of course vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

As used herein, " nab " stands for nanoparticle albumin bound, and "nanoparticle albumin bound sirolimus" is an albumin-stable nanoparticle formulation of sirolimus (rapamycin). Nanoparticle albumin bound sirolimus is also known as nanoparticle albumin bound rapamycin, which has been described previously. See, e.g., WO2008109163A1, WO2014151853, WO2008137148A2, and WO2012149451A1, each of which is incorporated herein by reference in its entirety.

As used herein, "treatment" or "treating" is a method for obtaining beneficial or desired results (including clinical results). For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, reducing the recurrence rate of the disease, delaying or slowing the progression of the disease, improving the disease state, alleviating the disease (partial or total), reducing the dosage of one or more other drugs required to treat the disease, delaying the progression of the disease, improving the quality of life and/or prolonging survival. In some embodiments, treatment reduces the severity of one or more symptoms associated with cancer by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to the corresponding symptoms in the same individual before treatment or compared to the corresponding symptoms in other individuals not receiving treatment. "Treatment" also encompasses a reduction in pathological findings of cancer. The methods described herein encompass any one or more of these treatment aspects.

As used herein, an individual "at risk" is an individual who is at risk for developing cancer. An individual "at risk" may or may not have had a detectable disease prior to treatment as described herein, and may or may not have demonstrated detectable disease. "At risk" means that the individual has one or more so-called risk factors, which are measurable parameters described herein that are associated with developing cancer. An individual who has one or more of these risk factors has a higher chance of developing cancer than an individual who does not have these one or more risk factors.

As used herein, "delaying" the onset of cancer means delaying, impeding, slowing, arresting, stabilizing and/or postponing the onset of the disease. This delay may be of varying lengths of time, depending on the disease being treated and/or the individual's medical history. As will be apparent to one skilled in the art, a sufficient or significant delay may actually encompass prevention, such that the individual would not develop the disease. A method for "delaying" the onset of cancer is one that reduces the chance of developing the disease within a given time frame and/or reduces the severity of the disease within a given time frame, compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of individuals. Cancer prevalence may be detected using standard methods including, but not limited to, computerized tomography (CAT scan), magnetic resonance imaging (MRI), ultrasound, coagulation tests, arteriovenous imaging, biopsy, urine cytology, and cystoscopy. Prevalence may also refer to the progression of cancer that may not initially be detectable and includes emergence, recurrence, and relapse.

The term "effective amount" as used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition or disease, such as to improve, alleviate, reduce and/or delay one or more of its symptoms. In the case of cancer, an effective amount includes an amount sufficient to shrink a tumor and/or reduce the rate of tumor growth (thereby curbing tumor growth) or to prevent or delay other unwanted cell proliferation in cancer. In some embodiments, an effective amount is an amount sufficient to delay the onset of cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. In some embodiments, an effective amount is an amount sufficient to reduce the recurrence rate in an individual. An effective amount can be administered in one or more administrations. An effective amount of the drug or composition can: (i) reduce the number of cancer cells; (ii) reduce the size of tumors; (iii) inhibit, delay, slow down and preferably stop the infiltration of cancer cells into peripheral organs to a certain extent; (iv) inhibit (i.e., slow down and preferably stop to a certain extent) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the appearance and/or recurrence of tumors; (vii) reduce the recurrence rate of tumors; and/or (viii) relieve to a certain extent one or more symptoms associated with cancer.

As understood in the art, an "effective amount" can be one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired therapeutic indicator. An effective amount can be considered in the context of administering one or more therapeutic agents, and a nanoparticle composition (e.g., a composition including sirolimus and albumin) can be considered to be administered in an effective amount if the desired or beneficial result can be achieved or achieved in combination with one or more other agents. The components of the combination therapy described herein (e.g., the first and second therapy) can be administered sequentially, simultaneously, or concurrently using the same or different routes of administration for each component. Thus, an effective amount of the combination therapy includes an amount of the first therapy and an amount of the second therapy that produce the desired result when administered sequentially, simultaneously, or concurrently.

"In conjunction with" or "in combination with" means administering a therapeutic modality in addition to another therapeutic modality, such as administering a nanoparticle composition described herein in addition to other agents to the same subject under the same treatment regimen. Thus, "in conjunction with" or "in combination with" means administering a therapeutic modality before, during, or after delivery of another therapeutic modality to a subject.

As used herein, the term "simultaneous administration" means that the first therapy and the second therapy in the combination therapy are administered with a time interval of no more than about 15 minutes (e.g., no more than about any of 10 minutes, 5 minutes, or 1 minute). When the first and second therapies are administered simultaneously, the first and second therapies may be contained in the same composition (e.g., a composition comprising both the first and second therapies) or in separate compositions (e.g., the first therapy is contained in one composition and the second therapy is contained in another composition).

As used herein, the term "sequential administration" means that the first therapy and the second therapy in the combination therapy are administered with a time interval of more than about 15 minutes (such as more than about 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes or more). The first therapy or the second therapy can be administered first. The first and second therapies are contained in separate compositions, which can be contained in the same or different packages or kits.

As used herein, the term "concurrent administration" means that the administration of a first therapy and the administration of a second therapy in the combination therapy overlap each other.

As used herein, the term "subject" for therapeutic purposes refers to any animal classified as follows: mammals including humans, domestic and farm animals, and zoo, sport or pet animals such as dogs, horses, cats, cows, etc. The mammal is preferably a human.

As used herein, "specificity", "specificity" or "selectivity" or "selectivity" when describing a compound as an inhibitor means that the compound interacts with (e.g., binds to, modulates and inhibits) a specific target (e.g., proteins and enzymes) better than non-targets. For example, the compound has a higher affinity, higher binding, higher binding coefficient or lower dissociation coefficient for a specific target. The specificity or selectivity of a compound for a specific target can be measured, determined or evaluated by using various methods well known in the art. For example, the specificity or selectivity can be measured, determined or evaluated by measuring the IC50 of the compound for the target. A compound is specific or selective for a target when its IC50 for a target is 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold or more lower than the IC50 of the same compound for a non-target. For example, the IC50 of a KRAS G12C inhibitor is 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold or more lower than the IC50 of the same KRAS G12C inhibitor for wild-type KRAS. IC50 can be determined by methods generally known in the art.

As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that is biologically or otherwise non-adverse, i.e., the material can be incorporated into a pharmaceutical composition for administration to a patient without causing any significant adverse biological effect or interacting in a deleterious manner with any other component of the composition in which it is contained. Pharmaceutically acceptable carriers or formulations preferably meet the required standards for toxicology and manufacturing testing and/or are included in the Inactive Ingredient Guide established by the U.S. Food and Drug Administration.

It should be understood that aspects and embodiments of the present invention include “comprising” aspects and embodiments, “consisting of” aspects and embodiments, and/or “consisting essentially of” aspects and embodiments.

As used herein, reference to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, description referring to "about X" includes description of "X".

As used herein, reference to "not being" a value or parameter generally means and describes "except for a value or parameter." For example, a method not being used to treat type X cancer means that the method is used to treat cancers of types other than type X.

As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly indicates otherwise. Methods of treating cancer

In some embodiments, a method of treating cancer in an individual (e.g., a human) is provided, comprising administering to the individual (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (e.g., a limus drug, such as sirolimus or a derivative or analog thereof) and albumin ("mTOR inhibitor nanoparticle composition"); and (b) an effective amount of a KRAS inhibitor (e.g., a KRAS G12C inhibitor, a KRAS G12A inhibitor, a KRAS G12D inhibitor, a KRAS G12F inhibitor, a KRAS G12L inhibitor, a KRAS G12R inhibitor, a KRAS G12S inhibitor, a KRAS G12V inhibitor, a KRAS G13A inhibitor, a KRAS G13C inhibitor, a KRAS G13A inhibitor, a KRAS G13B inhibitor, a KRAS G13C inhibitor, a KRAS G13C inhibitor, a KRAS G13C inhibitor, a KRAS G13A ... KRAS G13D inhibitor, KRAS G13P inhibitor, KRAS G13R inhibitor, KRAS G13S inhibitor, KRAS G13V inhibitor, KRAS Q61E inhibitor, KRAS Q61H inhibitor, KRAS Q61K inhibitor, KRAS Q61L inhibitor, KRAS Q61P inhibitor, KRAS Q61R inhibitor, KRAS K117N inhibitor, KRAS K117R inhibitor, KRAS A146E inhibitor, KRAS A146G inhibitor, KRAS A146P inhibitor, KRAS A146S inhibitor, KRAS A146T inhibitor or KRAS A146V inhibitor). In some embodiments, the cancer comprises a cell line that expresses a KRAS mutant protein (e.g., a KRAS G12C mutant protein, a KRAS G12A mutant protein, a KRAS G12D mutant protein, a KRAS G12F mutant protein, a KRAS G12L mutant protein, a KRAS G12R mutant protein, a KRAS G12S mutant protein, a KRAS G12V mutant protein, a KRAS G13A mutant protein, a KRAS G13C mutant protein, a KRAS G13D mutant protein, a KRAS G13P mutant protein, a KRAS G13R mutant protein, a KRAS G13S mutant protein, a KRAS G13V mutant protein, a KRAS Q61E mutant protein, a KRAS Q61H mutant protein, a KRAS Q61K mutant protein, a KRAS Q61L mutant protein, a KRAS Q61P mutant protein, a KRAS In some embodiments, the cancer comprises one or more cells having at least one mTOR activation aberration. In some embodiments, the cancer comprises one or more cells having at least one mTOR activation aberration. In some embodiments, the cancer is a solid tumor, lung cancer, bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, uterine cancer, endometrial cancer, cervical cancer, testicular cancer, bile duct cancer, myelodysplastic cancer, or a tumor of unknown origin. In some embodiments, the cancer or tumor (such as any of the aforementioned cancers or tumors) is advanced, unresectable and/or metastatic. In some embodiments, the cancer is a solid tumor (e.g., an advanced, unresectable and/or metastatic solid tumor), lung cancer (e.g., advanced, unresectable and/or metastatic lung cancer), or bladder cancer (e.g., advanced, unresectable and/or metastatic bladder cancer). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), such as advanced, unresectable and/or metastatic NSCLC.

In some embodiments, a method of treating cancer in an individual (e.g., a human) is provided, comprising administering to the individual (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (e.g., a limus drug, such as sirolimus or a derivative or analog thereof) and albumin ("mTOR inhibitor nanoparticle composition"), wherein the mTOR inhibitor in the nanoparticles is associated with the albumin (e.g., coated with the albumin); and (b) an effective amount of a KRAS inhibitor (e.g., a KRAS G12C inhibitor, a KRAS G12A inhibitor, a KRAS G12D inhibitor, a KRAS G12F inhibitor, a KRAS G12L inhibitor, a KRAS G12R inhibitor, a KRAS G12S inhibitor, a KRAS G12V inhibitor, a KRAS G12C inhibitor, a KRAS G12A inhibitor, a KRAS G12D inhibitor, a KRAS G12F inhibitor, a KRAS G12L inhibitor, a KRAS G12R inhibitor, a KRAS G12S inhibitor, a KRAS G12V inhibitor, a KRAS G12 G13A inhibitor, KRAS G13C inhibitor, KRAS G13D inhibitor, KRAS G13P inhibitor, KRAS G13R inhibitor, KRAS G13S inhibitor, KRAS G13V inhibitor, KRAS Q61E inhibitor, KRAS Q61H inhibitor, KRAS Q61K inhibitor, KRAS Q61L inhibitor, KRAS Q61P inhibitor, KRAS Q61R inhibitor, KRAS K117N inhibitor, KRAS K117R inhibitor, KRAS A146E inhibitor, KRAS A146G inhibitor, KRAS A146P inhibitor, KRAS A146S inhibitor, KRAS A146T inhibitor or KRAS A146V inhibitor). In some embodiments, the method comprises administering to a subject (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (e.g., a limus drug, such as sirolimus or a derivative or analog thereof) and albumin ("mTOR inhibitor nanoparticle composition"), wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm); and (b) an effective amount of a KRAS inhibitor (e.g., a KRAS G12C inhibitor, a KRAS G12A inhibitor, a KRAS G12D inhibitor, a KRAS G12F inhibitor, a KRAS G12L inhibitor, a KRAS G12R inhibitor, a KRAS G12S inhibitor, a KRAS G12V inhibitor, a KRAS G13A inhibitor, a KRAS G14 inhibitor, a KRAS G15A inhibitor, a KRAS G16 inhibitor, a KRAS G17 inhibitor, a KRAS G18 inhibitor, a KRAS G19 inhibitor, a KRAS G20 inhibitor, a KRAS G21 inhibitor, a KRAS G22A inhibitor, a KRAS G22D inhibitor, a KRAS G23 inhibitor, a KRAS G24 inhibitor, a KRAS G25 inhibitor, a KRAS G26 inhibitor, a KRAS G27 inhibitor, a KRAS G28 inhibitor, a KRAS G29 inhibitor, a KRAS G30 inhibitor, a KRAS G31 inhibitor, a KRAS G32 inhibitor, a KRAS G33 inhibitor, a KRAS G34 inhibitor, a KRAS G35 inhibitor, a KRAS G36 inhibitor, a KRAS G37 inhibitor, a KRAS G38 inhibitor, a KRAS G39 inhibitor, a KRAS G40 inhibitor, a KRAS G41 inhibitor, a KRAS G42 inhibitor, a KRAS G43 inhibitor, a KRAS G44 inhibitor, a KRA KRAS G13C inhibitor, KRAS G13D inhibitor, KRAS G13P inhibitor, KRAS G13R inhibitor, KRAS G13S inhibitor, KRAS G13V inhibitor, KRAS Q61E inhibitor, KRAS Q61H inhibitor, KRAS Q61K inhibitor, KRAS Q61L inhibitor, KRAS Q61P inhibitor, KRAS Q61R inhibitor, KRAS K117N inhibitor, KRAS K117R inhibitor, KRAS A146E inhibitor, KRAS A146G inhibitor, KRAS A146P inhibitor, KRAS A146S inhibitor, KRAS A146T inhibitor or KRAS A146V inhibitor). In some embodiments, the method comprises administering to a subject (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (e.g., a limus drug, such as sirolimus or a derivative or analog thereof) and albumin ("mTOR inhibitor nanoparticle composition"), wherein the nanoparticles comprise an mTOR inhibitor associated with albumin (e.g., coated with albumin), and wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm); and (b) an effective amount of a KRAS inhibitor (e.g., a KRAS G12C inhibitor, a KRAS G12A inhibitor, a KRAS G12D inhibitor, a KRAS G12F inhibitor, a KRAS G12L inhibitor, a KRAS G12R inhibitor, a KRAS G12 G12S inhibitors, KRAS G12V inhibitors, KRAS G13A inhibitors, KRAS G13C inhibitors, KRAS G13D inhibitors, KRAS G13P inhibitors, KRAS G13R inhibitors, KRAS G13S inhibitors, KRAS G13V inhibitors, KRAS Q61E inhibitors, KRAS Q61H inhibitors, KRAS Q61K inhibitors, KRAS Q61L inhibitors, KRAS Q61P inhibitors, KRAS Q61R inhibitors, KRAS K117N inhibitors, KRAS K117R inhibitors, KRAS A146E inhibitors, KRAS A146G inhibitors, KRAS A146P inhibitors, KRAS A146S inhibitor, KRAS A146T inhibitor, or KRAS A146V inhibitor). In some embodiments, the method comprises administering to a subject (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) and albumin ("mTOR inhibitor nanoparticle composition"), wherein the nanoparticles comprise an mTOR inhibitor associated with albumin (e.g., coated with albumin), wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm, e.g., about 100 nm), and wherein the weight ratio of albumin to mTOR inhibitor in the mTOR inhibitor nanoparticle composition is about 10:1 or less (e.g., about 10:1 or about 9:1 or about 8:1); and (b) an effective amount of a KRAS G12C inhibitor. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the mTOR inhibitor is sirolimus (rapamycin) or a derivative or analog thereof. In some embodiments, the mTOR inhibitor nanoparticle composition comprises nanoparticle albumin-bound sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is nanoparticle albumin-bound sirolimus. Exemplary mTOR inhibitor nanoparticle compositions that can be used with the methods provided herein are described in further detail below. In some embodiments, the cancer comprises a cell line that expresses a KRAS mutant protein (e.g., a KRAS G12C mutant protein, a KRAS G12A mutant protein, a KRAS G12D mutant protein, a KRAS G12F mutant protein, a KRAS G12L mutant protein, a KRAS G12R mutant protein, a KRAS G12S mutant protein, a KRAS G12V mutant protein, a KRAS G13A mutant protein, a KRAS G13C mutant protein, a KRAS G13D mutant protein, a KRAS G13P mutant protein, a KRAS G13R mutant protein, a KRAS G13S mutant protein, a KRAS G13V mutant protein, a KRAS Q61E mutant protein, a KRAS Q61H mutant protein, a KRAS Q61K mutant protein, a KRAS Q61L mutant protein, a KRAS Q61P mutant protein, a KRAS In some embodiments, the cancer comprises one or more cells having at least one mTOR activating aberration. Exemplary cancers treated according to the methods described herein are described elsewhere herein.

In some embodiments, a KRAS inhibitor (e.g., a KRAS G12C inhibitor, a KRAS G12A inhibitor, a KRAS G12D inhibitor, a KRAS G12F inhibitor, a KRAS G12L inhibitor, a KRAS G12R inhibitor, a KRAS G12S inhibitor, a KRAS G12V inhibitor, a KRAS G13A inhibitor, a KRAS G13C inhibitor, a KRAS G13D inhibitor, a KRAS G13P inhibitor, a KRAS G13R inhibitor, a KRAS G13S inhibitor, a KRAS G13V inhibitor, a KRAS Q61E inhibitor, a KRAS Q61H inhibitor, a KRAS Q61K inhibitor, a KRAS A KRAS Q61L inhibitor, a KRAS Q61P inhibitor, a KRAS Q61R inhibitor, a KRAS K117N inhibitor, a KRAS K117R inhibitor, a KRAS A146E inhibitor, a KRAS A146G inhibitor, a KRAS A146P inhibitor, a KRAS A146S inhibitor, a KRAS A146T inhibitor or a KRAS A146V inhibitor) is, for example, an inhibitor of a KRAS mutant protein (e.g., a KRAS G12C mutant protein, a KRAS G12A mutant protein, a KRAS G12D mutant protein, a KRAS G12F mutant protein, a KRAS G12L mutant protein, a KRAS G12R mutant protein, a KRAS G12S mutant protein, a KRAS G12V mutant protein, a KRAS KRAS G13A mutant protein, KRAS G13C mutant protein, KRAS G13D mutant protein, KRAS G13P mutant protein, KRAS G13R mutant protein, KRAS G13S mutant protein, KRAS G13V mutant protein, KRAS Q61E mutant protein, KRAS Q61H mutant protein, KRAS Q61K mutant protein, KRAS Q61L mutant protein, KRAS Q61P mutant protein, KRAS Q61R mutant protein, KRAS K117N mutant protein, KRAS K117R mutant protein, KRAS A146E mutant protein, KRAS A146G mutant protein, KRAS A146P mutant protein, KRAS A146S mutant protein, KRAS A146T mutant protein or KRAS A146V mutant protein) by targeting a polypeptide (such as an antibody), peptide, antisense oligonucleotide or small molecule.

Additional information about exemplary agents that can be used in the treatment of cancer can be found, for example, at www.mycancergenome.org/content/biomarkers/. Cancers comprising cells expressing a KRAS mutant protein (e.g., a KRAS G12C mutant protein, a KRAS G12A mutant protein, a KRAS G12D mutant protein, a KRAS G12F mutant protein, a KRAS G12L mutant protein, a KRAS G12R mutant protein, a KRAS G12S mutant protein, a KRAS G12V mutant protein, a KRAS G13A mutant protein, a KRAS G13C mutant protein, a KRAS G13D mutant protein, a KRAS G13P mutant protein, a KRAS G13R mutant protein, a KRAS G13S mutant protein, a KRAS G13V mutant protein, a KRAS Q61E mutant protein, a KRAS KRAS A146E mutant protein, KRAS A146G mutant protein, KRAS A146P mutant protein, KRAS A146S mutant protein, KRAS A146T mutant protein or KRAS A146V mutant protein) or more cells.

In some embodiments, the KRAS inhibitor is a KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is a small molecule. Exemplary small molecule KRAS G12C inhibitors that can be used with the methods provided herein include, but are not limited to, for example, sotolacib, also known as AMG 510 (Amgen/Beiji Shenzhou); MRTX849, also known as adagracib (Mirati/Zai Lab), JAB-21822 (Jacob Pharmaceuticals), GDC-6036 (Genentech), JDQ443 (Novartis), D-1553 (Invitrogen and Merck), GH35 (Qinhao Pharmaceuticals), GFH925 (Jinfang Pharmaceuticals), BPI-421286 (Beida Pharmaceuticals), LY3537982, RMC-6291 (Revolution Medicine), RMC-8839 (Revolution Medicine), HBI-2438 (Shanghai Asia Bioscience International Co., Ltd.) and JNJ-74699157 (Johnson & Johnson). Exemplary small molecule KRAS G12D inhibitors that can be used with the methods provided herein include, but are not limited to, MRTX1133 (Mirati Therapeutics) and RMC-6236 (Revolution Medicines). Exemplary small molecule KRAS G12V inhibitors that can be used with the methods provided herein include, but are not limited to, JAB-23000. Additional details about these and other exemplary KRAS inhibitors are described in further detail below. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS inhibitor (e.g., KRAS G12C inhibitor, KRAS G12A inhibitor, KRAS G12D inhibitor, KRAS G12F inhibitor, KRAS G12L inhibitor, KRAS G12R inhibitor, KRAS G12S inhibitor, KRAS G12V inhibitor, KRAS G13A inhibitor, KRAS G13C inhibitor, KRAS G13D inhibitor, KRAS G13P inhibitor, KRAS G13R inhibitor, KRAS G13S inhibitor, KRAS G13V inhibitor, KRAS Q61E inhibitor, KRAS Q61H inhibitor, KRAS In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS inhibitor are administered sequentially (i.e., the administration periods of the mTOR inhibitor nanoparticle composition and the KRAS inhibitor do not overlap with each other). In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS inhibitor are administered simultaneously. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS inhibitor are administered concurrently (ie, the administration periods of the mTOR inhibitor nanoparticle composition and the KRAS inhibitor overlap with each other).

In some embodiments, a method of treating cancer in an individual (e.g., a human) is provided, comprising administering to the individual (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (e.g., a limus drug, such as sirolimus or a derivative or analog thereof) and albumin ("mTOR inhibitor nanoparticle composition"); and (b) an effective amount of a KRAS inhibitor (e.g., a KRAS G12C inhibitor, a KRAS G12A inhibitor, a KRAS G12D inhibitor, a KRAS G12F inhibitor, a KRAS G12L inhibitor, a KRAS G12R inhibitor, a KRAS G12S inhibitor, a KRAS G12V inhibitor, a KRAS G13A inhibitor, a KRAS G13C inhibitor, a KRAS G13A inhibitor, a KRAS G13B inhibitor, a KRAS G13C inhibitor, a KRAS G13C inhibitor, a KRAS G13C inhibitor, a KRAS G13A ... G13D inhibitor, KRAS G13P inhibitor, KRAS G13R inhibitor, KRAS G13S inhibitor, KRAS G13V inhibitor, KRAS Q61E inhibitor, KRAS Q61H inhibitor, KRAS Q61K inhibitor, KRAS Q61L inhibitor, KRAS Q61P inhibitor, KRAS Q61R inhibitor, KRAS K117N inhibitor, KRAS K117R inhibitor, KRAS A146E inhibitor, KRAS A146G inhibitor, KRAS A146P inhibitor, KRAS A146S inhibitor, KRAS A146T inhibitor, or KRAS A146V inhibitor), wherein the mTOR inhibitor nanoparticle composition and the KRAS inhibitor are administered concurrently. In some embodiments, the cancer comprises a cell line that expresses a KRAS mutant protein (e.g., a KRAS G12C mutant protein, a KRAS G12A mutant protein, a KRAS G12D mutant protein, a KRAS G12F mutant protein, a KRAS G12L mutant protein, a KRAS G12R mutant protein, a KRAS G12S mutant protein, a KRAS G12V mutant protein, a KRAS G13A mutant protein, a KRAS G13C mutant protein, a KRAS G13D mutant protein, a KRAS G13P mutant protein, a KRAS G13R mutant protein, a KRAS G13S mutant protein, a KRAS G13V mutant protein, a KRAS Q61E mutant protein, a KRAS Q61H mutant protein, a KRAS Q61K mutant protein, a KRAS Q61L mutant protein, a KRAS Q61P mutant protein, a KRAS In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS inhibitor are administered at about the same time (e.g., within 1, 2, 3, 4, 5, 6, or 7 days). In some embodiments, administration of the mTOR inhibitor nanoparticle composition and the KRAS inhibitor are terminated at about the same time (e.g., within any of 1, 2, 3, 4, 5, 6, or 7 days). In some embodiments, administration of the KRAS inhibitor continues after administration of the mTOR inhibitor nanoparticle composition is terminated (e.g., any of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months). In some embodiments, administration of the KRAS inhibitor is initiated after administration of the mTOR inhibitor nanoparticle composition is initiated (e.g., after any of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months). In some embodiments, administration of the mTOR inhibitor nanoparticle composition and the KRAS inhibitor is initiated and terminated at about the same time. In some embodiments, administration of the mTOR inhibitor nanoparticle composition and the KRAS inhibitor is initiated at about the same time, and administration of the KRAS inhibitor continues after administration of the mTOR nanoparticle composition is terminated (e.g., any of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months). In some embodiments, administration of the mTOR nanoparticle composition and the KRAS inhibitor is stopped at about the same time, and administration of the KRAS inhibitor is initiated after administration of the mTOR inhibitor nanoparticle composition is initiated (e.g., after any of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months). In some embodiments, administration of the nanoparticle composition and the KRAS inhibitor is stopped at about the same time, and administration of the mTOR inhibitor nanoparticle composition is initiated after administration of the KRAS inhibitor is initiated (e.g., after any of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months).

In some embodiments of any of the methods described herein, the individual (e.g., human) has been diagnosed with or is suspected of having cancer. In some embodiments, the cancer comprises one or more cells expressing a KRAS mutant protein. Additionally or alternatively, in some embodiments, the cancer comprises one or more cells having at least one mTOR activation aberration. In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc.

In some embodiments of any of the methods described herein, the method comprises (e.g., further comprises) administering an mTOR inhibitor nanoparticle composition and a KRAS inhibitor (e.g., a KRAS G12C inhibitor, a KRAS G12A inhibitor, a KRAS G12D inhibitor, a KRAS G12F inhibitor, a KRAS G12L inhibitor, a KRAS G12R inhibitor, a KRAS G12S inhibitor, a KRAS G12V inhibitor, a KRAS G13A inhibitor, a KRAS G13C inhibitor, a KRAS G13D inhibitor, a KRAS G13P inhibitor, a KRAS G13R inhibitor, a KRAS G13S inhibitor, a KRAS G13V inhibitor, a KRAS Q61E inhibitor, a KRAS In some embodiments, the subject is selected for treatment based on the presence of one or more cancer cells having at least one mTOR activating aberration in a sample from the subject prior to administration of a KRAS Q61H inhibitor, a KRAS Q61K inhibitor, a KRAS Q61L inhibitor, a KRAS Q61P inhibitor, a KRAS Q61R inhibitor, a KRAS K117N inhibitor, a KRAS K117R inhibitor, a KRAS A146E inhibitor, a KRAS A146G inhibitor, a KRAS A146P inhibitor, a KRAS A146S inhibitor, a KRAS A146T inhibitor, or a KRAS A146V inhibitor. In some embodiments, the mTOR activating aberration comprises a mutation in an mTOR-related gene. In some embodiments, the mTOR activation aberration comprises a change in the number of copies of an mTOR-related gene. In some embodiments, the mTOR activation aberration comprises an abnormal expression of an mTOR-related gene. In some embodiments, the mTOR activation aberration comprises an abnormal activity level of an mTOR-related gene. In some embodiments, at least one mTOR activation aberration comprises an abnormal phosphorylation level of a protein encoded by an mTOR-related gene. In some embodiments, the mTOR activation aberration is in at least one mTOR-related gene selected from the group consisting of: AKT1, FLT-3, MTOR, PIK3CA, PIK3CG, TSC1, TSC2, RHEB, STK11, NF1, NF2, TP53, FGFR4, BAP1, KRAS, NRAS, NRF2, KEAP1 and PTEN. In some embodiments, at least one mTOR-related gene is TSC1 and/or TSC2. In some embodiments, mTOR activation aberrations are assessed (e.g., detected) by gene sequencing (e.g., next generation sequencing or "NGS"). In some embodiments, mTOR activation aberrations are assessed (e.g., detected) by sequencing DNA in a tumor sample (e.g., a formalin-fixed paraffin-embedded tumor sample) from an individual. In some embodiments, mTOR activation aberrations are assessed (e.g., detected) by sequencing circulating or free DNA in a blood sample from an individual. Additional details regarding mTOR activation aberrations and assessing/detecting mTOR activation aberrations in samples obtained from individuals with cancer (e.g., tumor samples or blood samples) are described below and in WO 2017/004267, the contents of which are incorporated herein by reference in their entirety.

In some embodiments of any of the methods described herein, the method comprises (e.g., further comprises) determining, prior to administering the mTOR inhibitor nanoparticle composition and the KRAS inhibitor, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, whether or not the mTOR inhibitor is present in the sample from the individual, In some embodiments, the present invention selects an individual for treatment by detecting the presence of one or more cancer cells of a KRAS mutant protein (e.g., KRAS Q61H mutant protein, KRAS Q61K mutant protein, KRAS Q61L mutant protein, KRAS Q61P mutant protein, KRAS Q61R mutant protein, KRAS K117N mutant protein, KRAS K117R mutant protein, KRAS A146E mutant protein, KRAS A146G mutant protein, KRAS A146P mutant protein, KRAS A146S mutant protein, KRAS A146T mutant protein, or KRAS A146V mutant protein). In some embodiments, the KRAS mutation is assessed (e.g., detected) by genetic sequencing (e.g., next generation sequencing or "NGS"). In some embodiments, KRAS mutations are assessed (e.g., detected) by sequencing DNA in a cancer sample (e.g., a formalin-fixed paraffin-embedded cancer sample) from an individual. In some embodiments, KRAS mutations are assessed (e.g., detected) by sequencing circulating or cell-free DNA in a blood sample from an individual.

For example, cancer treatment can be evaluated by tumor regression, tumor weight or size reduction, time to progression (TTP), duration of survival (DOS), progression-free survival (PFS), overall survival (OS), objective response rate (ORR), duration of response (DOR), quality of life (QoL), protein expression and/or activity. Methods for determining efficacy of treatments can be employed, including, for example, measuring responses via radiographic imaging. In some embodiments, treatment efficacy is measured as percent tumor growth inhibition (TGI %), which is calculated using the formula 100×(ΔC-ΔT)/ΔC, where ΔT and ΔC are the change in mean tumor volume between the last day that all animals in the saline or control groups survived and the first day of measurement for the treatment and control groups, respectively. In some embodiments, TGI% is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95% or more than 95% (e.g., more than about 96%, 97%, 98% or 99%). In some embodiments, overall survival (OS) is defined as the time from the start of self-treatment to the date of death from any cause. In some embodiments, progression-free survival (PFS) is defined as the time from the start of self-treatment to the date of disease progression (PD) or death from any cause, whichever comes first. In some embodiments, duration of response (DOR) is defined as the time from the start of autonomous treatment to the first documented objective tumor response (complete response ("CR") or partial response ("PR")) to the first documented PD or death from any cause, whichever comes first. In some embodiments, objective response rate (ORR) is defined as the proportion of individuals (e.g., patients, individuals) who experience a confirmed complete response (CR) or partial response (PR) based on RECIST v1.1 criteria during the period from the first treatment dose to the last treatment dose. Composition comprising nanoparticles comprising an mTOR inhibitor and albumin mTOR inhibitor

The methods provided herein comprise administering to a subject having cancer an effective amount of an mTOR inhibitor nanoparticle composition. In some embodiments, the cancer comprises a cell line that expresses a KRAS mutant protein (e.g., a KRAS G12C mutant protein, a KRAS G12A mutant protein, a KRAS G12D mutant protein, a KRAS G12F mutant protein, a KRAS G12L mutant protein, a KRAS G12R mutant protein, a KRAS G12S mutant protein, a KRAS G12V mutant protein, a KRAS G13A mutant protein, a KRAS G13C mutant protein, a KRAS G13D mutant protein, a KRAS G13P mutant protein, a KRAS G13R mutant protein, a KRAS G13S mutant protein, a KRAS G13V mutant protein, a KRAS Q61E mutant protein, a KRAS Q61H mutant protein, a KRAS Q61K mutant protein, a KRAS Q61L mutant protein, a KRAS Q61P mutant protein, a KRAS In some embodiments, the invention relates to a method for treating a cancer cell that is at least one of a plurality of mTOR-activating aberrations and a plurality of mTOR-activating aberrations. In some embodiments, the invention relates to a method for treating a cancer cell that is at least one of a plurality of mTOR-activating aberrations and a plurality of mTOR-activating aberrations. In some embodiments, the invention relates to a method for treating a cancer cell that is at least one of a plurality of mTOR-activating aberrations and a plurality of mTOR-activating aberrations. In some embodiments, the invention relates to a method for treating a cancer cell that is at least one of a plurality of mTOR-activating aberrations and a plurality of mTOR-activating aberrations. In some embodiments, the invention relates to a method for treating a cancer cell that is at least one of a plurality of mTOR-activating aberrations and a plurality of mTOR-activating aberrations. mTOR is a serine/threonine-specific protein kinase downstream of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) pathway and is a key regulator of cell survival, proliferation, stress, and metabolism. Dysregulation of the mTOR pathway has been found in many human carcinomas, and mTOR inhibition produces a significant inhibitory effect on tumor progression.

Mammalian target of rapamycin (mTOR) (also known as mechanistic target of rapamycin or FK506 binding protein 12-rapamycin-associated protein 1 (FRAP1)) is an atypical serine/threonine protein kinase present in two different complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is composed of mTOR, regulatory-associated protein of mTOR (Raptor), mammalian lethal SEC13 protein 8 (MLST8), PRAS40 and DEPTOR (Kim et al. (2002), Cell 110:163-75; Fang et al. (2001), Science 294 (5548):1942-5). mTORC1 integrates four major signaling inputs: nutrients (such as amino acids and phosphatidic acid), growth factors (insulin), energy, and stress (such as hypoxia and DNA damage). Amino acid availability signals to mTORC1 via pathways involving Rag and Ragulator (LAMTOR1-3) growth factors and hormones (such as insulin) signal to mTORC1 via Akt, which does not activate TSC2 to prevent mTORC1 inhibition. Instead, low ATP levels lead to AMPK-dependent activation of TSC2 and phosphorylation of raptor to reduce mTORC1 signaling proteins.

Active mTORC1 has multiple downstream biological effects, including activation of mRNA translation via phosphorylation of downstream targets (4E-BP1 and p70 S6 kinase), autophagy inhibition (Atg13, ULK1), ribosome biogenesis, and transcription leading to mitochondrial metabolism or lipogenesis. Thus, mTORC1 activity promotes cell growth when conditions are favorable or promotes catabolic processes during stress or when conditions are unfavorable.

mTORC2 is composed of mTOR, the rapamycin-insensitive partner of mTOR (RICTOR), GβL, and mammalian stress-activated protein kinase-interacting protein 1 (mSIN1). Compared to mTORC1, for which many upstream signals and cellular functions have been defined (see above), relatively little is known about the biology of mTORC2. mTORC2 regulates cytoskeletal organization through its stimulation of F-actin stress fibers, paxillin, RhoA, Rac1, Cdc42, and protein kinase Cα (PKCα). It has been observed that blocking the gene expression of mTORC2 components affects actin polymerization and interferes with cell morphology (Jacinto et al. (2004), Nat. Cell Biol. 6 , 1122-1128; Sarbassov et al. (2004), Curr. Biol. 14 , 1296-1302). This suggests that mTORC2 controls the actin cytoskeleton by promoting protein kinase Cα (PKCα) phosphorylation, staphylin phosphorylation and its relocalization to focal adhesions and GTP loading of RhoA and Rac1. The molecular mechanism by which mTORC2 regulates these processes has not yet been determined.

In some embodiments, the mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) is an inhibitor of mTORC1. In some embodiments, the mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) is an inhibitor of mTORC2. In some embodiments, the mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) is an inhibitor of both mTORC1 and mTORC2.

In some embodiments, the mTOR inhibitor is a limus drug, including sirolimus (also known as rapamycin and rapamycin) and its derivatives and analogs. Other exemplary limus drugs include, but are not limited to, temsirolimus (also known as CCI-779 and urosel), everolimus (also known as RAD001, Zortress, Certican and Afinitor), defostiolimus (AP-23573), deferolimus (MK-8669), zotarolimus (ABT-578), pimecrolimus and tacrolimus (FK-506). In some embodiments, the limus drug is selected from the group consisting of temsirolimus (CCI-779), everolimus (RAD001), dafolimus (AP-23573), deferolimus (MK-8669), zotarolimus (ABT-578), pimecrolimus and tacrolimus (FK-506). In some embodiments, the mTOR inhibitor is an mTOR kinase inhibitor, such as CC-115 or CC-223.

In some embodiments, the mTOR inhibitor is sirolimus (rapamycin). Sirolimus is a macrolide antibiotic that complexes with FKBP-12 and inhibits the mTOR pathway by binding to mTORC1.

Other exemplary mTOR inhibitors include, but are not limited to, BEZ235 (NVP-BEZ235), AZD8055, PI-103, Ku-0063794, INK 128, AZD2014, NVP-BGT226, PF-04691502, CH5132799, GDC-0980 (RG7422), Torin 1, WAY-600, WYE-125132, WYE-687, GSK2126458, PF-05212384 (PKI-587), PP-121, OSI-027, Palomid 529, PP242, XL765, GSK1059615, and WYE-354.

BEZ235 (NVP-BEZ235) is an imidazoquinoline derivative that is an inhibitor of mTORC1 catalysis (Roper J et al., PLoS One, 2011, 6(9), e25132). Everolimus is a 40-O-(2-hydroxyethyl) derivative of sirolimus and binds to the cyclophilin FKBP-12, and this complex is also mTORC1. AZD8055 is a small molecule that inhibits mTORC1 phosphorylation (p70S6K and 4E-BP1). Temsirolimus is a small molecule that forms a complex with FK506-binding protein and prevents mTOR activation when it is present in the mTORC1 complex. PI-103 is a small molecule that inhibits the activation of the rapamycin-sensitive (mTORC1) complex (Knight et al. (2006), Cell. 125: 733-47). KU-0063794 is a small molecule that inhibits phosphorylation of mTORC1 at Ser2448 in a dose-dependent and time-dependent manner. INK 128, AZD2014, NVP-BGT226, CH5132799, WYE-687 and each are small molecule inhibitors of mTORC1. PF-04691502 inhibits mTORC1 activity. GDC-0980 is an orally bioavailable small molecule that inhibits class I PI3 kinases and TORC1. Torin 1 is a potent small molecule inhibitor of mTOR. WAY-600 is a potent, ATP-competitive and selective inhibitor of mTOR. WYE-125132 is an ATP-competitive small molecule inhibitor of mTORC1. GSK2126458 is an inhibitor of mTORC1. PKI-587 is a highly potent dual inhibitor of PI3Kα, PI3Kγ, and mTOR. PP-121 is a multi-target inhibitor of PDGFR, Hck, mTOR, VEGFR2, Src, and Abl. OSI-027 is a selective and potent dual inhibitor of mTORC1 and mTORC2 with IC50 of 22 nM and 65 nM, respectively. Palomid 529 is a small molecule inhibitor of mTORC1 that has no affinity for ABCB1/ABCG2 and has good brain penetration (Lin et al. (2013), Int J Cancer DOI: 10.1002/ijc. 28126 (electronic version ahead of print)). PP242 is a selective mTOR inhibitor. XL765 is a dual inhibitor of mTOR/PI3k targeting mTOR, p110α, p110β, p110γ, and p110δ. GSK1059615 is a novel and dual inhibitor of PI3Kα, PI3Kβ, PI3Kδ, PI3Kγ, and mTOR. WYE-354 inhibits mTORC1 in Hek293 cells (0.2 μM-5 μM) and HUVEC cells (10 nM-1 μM). WYE-354 is a potent, specific, and ATP-competitive inhibitor of mTOR. Deferolimus (defolimus, AP23573, MK-8669) is a selective mTOR inhibitor.

In some embodiments, the mTOR inhibitor is a derivative or analog of any of the mTOR inhibitors described herein. In some embodiments, a "derivative" or "analog" of an mTOR inhibitor refers to a compound that is structurally similar to an mTOR inhibitor or is in the same general chemical class as an mTOR inhibitor. In some embodiments, a derivative or analog of an mTOR inhibitor retains similar chemical and/or physical properties (including, for example, functionality) of a second therapeutic agent or moiety.

In some embodiments, a method of treating cancer in an individual (e.g., a human) is provided, comprising administering to the individual (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin ("mTOR inhibitor nanoparticle composition"), wherein the mTOR inhibitor is selected from the group consisting of sirolimus (also known as rapamycin and rapamycin) or a derivative or analog thereof, temsirolimus (also known as CCI-779 and urosel), everolimus (also known as RAD001, Zortress, Certican and Afinitor), dafolimus (AP-23573), deferolimus (MK-8669), zotarolimus (ABT-578), pimecrolimus and tacrolimus (FK-506), BEZ235 (NVP-BEZ235), AZD8055, PI-103, Ku-0063794, INK 128, AZD2014, NVP-BGT226, PF-04691502, CH5132799, GDC-0980 (RG7422), Torin 1, WAY-600, WYE-125132, WYE-687, GSK2126458, PF-05212384 (PKI-587), PP-121, OSI-027, Palomid 529, PP242, XL765, GSK1059615 and WYE-354; and (b) an effective amount of a KRAS inhibitor (e.g., a KRAS G12C inhibitor, a KRAS G12A inhibitor, a KRAS KRAS G12D inhibitor, KRAS G12F inhibitor, KRAS G12L inhibitor, KRAS G12R inhibitor, KRAS G12S inhibitor, KRAS G12V inhibitor, KRAS G13A inhibitor, KRAS G13C inhibitor, KRAS G13D inhibitor, KRAS G13P inhibitor, KRAS G13R inhibitor, KRAS G13S inhibitor, KRAS G13V inhibitor, KRAS Q61E inhibitor, KRAS Q61H inhibitor, KRAS Q61K inhibitor, KRAS Q61L inhibitor, KRAS Q61P inhibitor, KRAS Q61R inhibitor, KRAS K117N inhibitor, KRAS In some embodiments, the mTOR inhibitor is sirolimus. In some embodiments, the cancer comprises a cell line that expresses a KRAS mutant protein (e.g., a KRAS G12C mutant protein, a KRAS G12A mutant protein, a KRAS G12D mutant protein, a KRAS G12F mutant protein, a KRAS G12L mutant protein, a KRAS G12R mutant protein, a KRAS G12S mutant protein, a KRAS G12V mutant protein, a KRAS G13A mutant protein, a KRAS G13C mutant protein, a KRAS G13D mutant protein, a KRAS G13P mutant protein, a KRAS G13R mutant protein, a KRAS G13S mutant protein, a KRAS G13V mutant protein, a KRAS Q61E mutant protein, a KRAS Q61H mutant protein, a KRAS Q61K mutant protein, a KRAS Q61L mutant protein, a KRAS Q61P mutant protein, a KRAS In some embodiments, the invention relates to a method for treating a cancerous cell or a tumor of unknown origin. The method comprises one or more cancer cells of a type 2 cancerous cell or a type 2 cancerous cell ... In some embodiments, the cancer or tumor (such as any of the aforementioned cancers or tumors) is advanced, unresectable and/or metastatic. In some embodiments, the cancer is a solid tumor (e.g., an advanced, unresectable and/or metastatic solid tumor), lung cancer (e.g., advanced, unresectable and/or metastatic lung cancer) or bladder cancer (e.g., advanced, unresectable and/or metastatic bladder cancer). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), such as advanced, unresectable and/or metastatic NSCLC. The mTOR inhibitor nanoparticle composition (such as sirolimus/albumin nanoparticle composition) can be administered via any of the accepted modes of administration or medicaments known in the art. In some embodiments, the mTOR inhibitor nanoparticle composition is administered intravenously or subcutaneously. In some embodiments, the mTOR inhibitor nanoparticle composition (such as sirolimus/albumin nanoparticle composition) and the KRAS inhibitor are administered in a single unit dose. In some embodiments, the mTOR inhibitor nanoparticle composition (such as sirolimus/albumin nanoparticle composition) and the KRAS inhibitor are administered in separate dosage forms.

In some embodiments, a method of treating cancer in an individual (e.g., a human) is provided, comprising administering to the individual (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor (e.g., sirolimus or a derivative or analog thereof) and albumin, and (b) a KRAS G12C inhibitor (e.g., sotolacib or adagraciib). In some embodiments, the nanoparticles are administered once a week, twice every three weeks (e.g., day 1 and day 8 of a 21-day cycle), or once every three weeks (e.g., intravenously or subcutaneously). In some embodiments, the amount of mTOR inhibitor (e.g., sirolimus) administered each time is about 1-75 mg/m ² . In some embodiments, the KRAS G12C inhibitor is administered once a day or twice a day (e.g., orally). In some embodiments, the amount of the KRAS G12C inhibitor administered each time is about 200-800 mg, where the KRAS G12C inhibitor is administered twice a day (e.g., 12 hours apart) as appropriate. In some embodiments, the amount of the KRAS G12C inhibitor administered each time is about 100-2000 mg, where the KRAS G12C inhibitor is administered once a day as appropriate. In some embodiments, the cancer is a locally advanced or metastatic cancer. In some embodiments, the cancer is a solid tumor (e.g., lung cancer, such as NSCLC, such as bladder cancer). In some embodiments, the cancer comprises an mTOR activation aberration in STK11, TP53, ATM, CDKN2A, or UGT2B17. In some embodiments, the cancer comprises mTOR activating aberrations in one or more genes selected from the group consisting of TP53, STK11, PTEN, ATM, CDKN2A, and UGT2B17. Nanoparticle Compositions

mTOR inhibitor nanoparticle compositions that can be used with the methods described herein include nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) and an albumin (such as human serum albumin, in various embodiments consisting essentially of or consisting of the same). Nanoparticles of poorly water-soluble drugs such as macrolides have been disclosed in, for example, U.S. Pat. Nos. 5,916,596, 6,506,405, 6,749,868, 6,537,579, 7,820,788, and 8,911,786, and U.S. Patent Publication Nos. 2006/0263434 and 2007/0082838; PCT Patent Application WO 08/137148, each of which is incorporated herein by reference in its entirety.

In some embodiments, the mTOR inhibitor nanoparticle composition comprises nanoparticles having an average diameter of no more than about 1000 nanometers (nm), such as no more than about any one of 900, 800, 700, 600, 500, 400, 300, 200, and 100 nm. In some embodiments, the average diameter of the nanoparticles is no more than about 200 nm. In some embodiments, the average diameter of the nanoparticles is no more than about 150 nm. In some embodiments, the average diameter of the nanoparticles is no more than about 100 nm. In some embodiments, the average diameter of the nanoparticles is about 10 to about 400 nm. In some embodiments, the average diameter of the nanoparticles is about 10 to about 150 nm. In some embodiments, the average diameter of the nanoparticles is about 40 to about 120 nm. In some embodiments, the nanoparticles are no smaller than about 50 nm. In some embodiments, the nanoparticles are sterile filterable.

In some embodiments, the average diameter of the nanoparticles in the mTOR inhibitor nanoparticle composition is no more than about 200 nm, including, for example, no more than about 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, or 60 nm. In some embodiments, at least about 50% (e.g., at least about 60%, 70%, 80%, 90%, 95%, or 99%) of the nanoparticles in the mTOR inhibitor nanoparticle composition have a diameter of no more than about 200 nm, including, for example, no more than about 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, or 60 nm. In some embodiments, at least about 50% (e.g., at least any of 60%, 70%, 80%, 90%, 95%, or 99%) of the nanoparticles in the mTOR inhibitor nanoparticle composition are within the range of about 10 nm to about 400 nm, including, for example, about 10 nm to about 200 nm, about 20 nm to about 200 nm, about 30 nm to about 180 nm, about 40 nm to about 150 nm, about 40 nm to about 120 nm, and about 60 nm to about 100 nm.

In some embodiments, the albumin in the mTOR inhibitor nanoparticle composition has sulfhydryl groups that can form disulfide bonds. In some embodiments, at least about 5% (including, for example, at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the albumin in the nanoparticle portion of the composition is cross-linked (e.g., via one or more disulfide bonds).

In some embodiments, nanoparticles comprising an mTOR inhibitor described herein (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) are associated with (e.g., coated with) an albumin (e.g., human albumin or human serum albumin). In some embodiments, the composition comprises an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) in both nanoparticle and non-nanoparticle form (e.g., in solution or in the form of a soluble albumin/nanoparticle complex), wherein at least about any one of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the mTOR inhibitor in the composition is in nanoparticle form. In some embodiments, the mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) in the nanoparticles comprises more than about any of 50%, 60%, 70%, 80%, 90%, 95%, or 99% by weight of the nanoparticles. In some embodiments, the nanoparticles have a non-polymeric matrix. In some embodiments, the nanoparticles comprise a core of an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) that is substantially free of polymeric material (such as a polymeric matrix).

In some embodiments, the mTOR inhibitor nanoparticle composition comprises albumin in both the nanoparticle and non-nanoparticle portions of the composition, wherein at least about any of 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the albumin in the composition is in the non-nanoparticle portion of the composition.

In some embodiments, the weight ratio of albumin (such as human albumin or human serum albumin) to mTOR inhibitor (such as limus drug, such as sirolimus or its derivative or analog) in the mTOR inhibitor nanoparticle composition is about 18: 1 or less, such as about 15: 1 or less, such as about 10: 1 or less. In some embodiments, the weight ratio of albumin (such as human albumin or human serum albumin) to mTOR inhibitor (such as limus drug, such as sirolimus or its derivative or analog) in the mTOR inhibitor nanoparticle composition is about 1: 1 to about 18: 1, about 2: 1 to about 15: 1, about 3: 1 to about 13: 1, about 4: 1 to about 12: 1, about 5: 1 to about 10: 1. In some embodiments, the weight ratio of albumin to mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) in the nanoparticle portion of the mTOR inhibitor nanoparticle composition is about any of 1:2, 1:3, 1:4, 1:5, 1:9, 1:10, 1:15 or less. In some embodiments, the weight ratio of albumin (such as human albumin or human serum albumin) to mTOR inhibitor (such as limus drugs, such as sirolimus or its derivatives or analogs) in the mTOR inhibitor nanoparticle composition is any one of the following: about 1:1 to about 18:1, about 1:1 to about 15:1, about 1:1 to about 12:1, about 1:1 to about 10:1, about 1:1 to about 9:1, about 1:1 to about 8:1, about 1:1 to about 7:1, about 1:1 to about 6:1, about 1:1 to about 5:1, about 1:1 to about 4:1, about 1:1 to about 3:1, about 1:1 to about 2:1, about 1:1 to about 1:1.

In some embodiments, the mTOR inhibitor nanoparticle composition (such as sirolimus/albumin nanoparticle composition) comprises one or more of the above characteristics.

In some embodiments, the mTOR inhibitor nanoparticle composition that can be used with the methods described herein is a dry formulation (such as a lyophilized composition). In some embodiments, the mTOR inhibitor nanoparticle composition is suspended in a biocompatible medium. Suitable biocompatible media include, but are not limited to: water, buffered aqueous media, saline, buffered saline, optionally buffered amino acid solutions, optionally buffered protein solutions, optionally buffered sugar solutions, optionally buffered vitamin solutions, optionally buffered synthetic polymer solutions, lipid-containing emulsions, and the like.

In some embodiments, the mTOR inhibitor nanoparticle composition comprises albumin (such as human albumin or human serum albumin). The albumin can be of natural origin or prepared synthetically. In some embodiments, the albumin is human albumin or human serum albumin. In some embodiments, the albumin is recombinant albumin.

Human serum albumin (HSA) is a highly soluble globular protein with Mr 65K and is composed of 585 amino acids. HSA is the most abundant protein in plasma and is responsible for 70%-80% of the colloidal osmotic pressure of human plasma. The amino acid sequence of HSA contains a total of 17 disulfide bridges, a free thiol (Cys 34) and a single tryptophan (Trp 214). Intravenous use of HSA solutions has been indicated for the prevention and treatment of hypovolemic shock (see, e.g., Tullis, JAMA, 237: 355-360, 460-463, (1977) and Houser et al., Surgery, Gynecology and Obstetrics, 150: 811-816 (1980)) and in combination with exchange transfusion for the treatment of neonatal hypercholesterolemia (see, e.g., Finlayson, Seminars in Thrombosis and Hemostasis, 6, 85-120, (1980)). Other albumins are contemplated, such as bovine serum albumin. The use of such non-human albumins may be appropriate, for example, in situations where such compositions are to be used in non-human mammals, such as in veterinary medicine (including domestic pets and agricultural situations). Human serum albumin (HSA) has multiple hydrophobic binding sites (eight in total for fatty acids, the endogenous ligands of HSA) and binds a wide variety of drugs, especially neutral and negatively charged hydrophobic compounds (Goodman et al., The Pharmacological Basis of Therapeutics, 9th edition, McGraw-Hill New York (1996)). Two high affinity binding sites have been proposed in subdomains IIA and IIIA of HSA, which are highly elongated hydrophobic pockets with charged lysine and arginine residues near the surface that serve as attachment points for polar ligand characteristics (see, e.g., Fehske et al., Biochem. Pharmcol., 30, 687-92 (198a); Vorum, Dan. Med. Bull., 46, 379-99 (1999); Kragh-Hansen, Dan. Med. Bull., 1441, 131-40 (1990); Curry et al., Nat. Struct. Biol., 5, 827-35 (1998); Sugio et al., Protein. Eng., 12, 439-46 (1999); He et al., Nature, 358, 359-47 (1999). 209-15 (199b); and Carter et al., Adv. Protein. Chem., 45, 153-203 (1994)). Rapamycin and propofol have been shown to bind to HSA (see, e.g., Paal et al., Eur. J. Biochem., 268(7), 2187-91 (200a); Purcell et al., Biochim. Biophys. Acta, 1478(a), 61-8 (2000); Altmayer et al., Arzneimittelforschung, 45, 1053-6 (1995); and Garrido et al., Rev. Esp. Anestestiol. Reanim., 41, 308-12 (1994)). Additionally, docetaxel has been shown to bind to human plasma proteins (see, e.g., Urien et al., Invest. New Drugs, 14(b), 147-51 (1996)).

The albumin (such as human albumin or human serum albumin) in the mTOR inhibitor nanoparticle composition generally acts as a carrier for the mTOR inhibitor, that is, the albumin in the composition allows the mTOR inhibitor (such as a limus drug, such as sirolimus or a derivative or analog thereof) to be more easily suspended in an aqueous medium or helps maintain the suspension compared to a composition that does not contain albumin. This can avoid the use of toxic solvents (or surfactants) for dissolving the mTOR inhibitor, and thereby reduce one or more side effects of administering an mTOR inhibitor (such as a limus drug, such as sirolimus or a derivative thereof) to an individual (such as a human). Thus, in some embodiments, the mTOR inhibitor nanoparticle compositions that can be used with the methods described herein are substantially free of (e.g., free of) surfactants, such as cetyl alcohol polyoxyethylene ether (or polyoxyethylated castor oil, including Cremophor EL® (BASF)). In some embodiments, the mTOR inhibitor nanoparticle compositions (e.g., sirolimus/albumin nanoparticle compositions) are substantially free of (e.g., free of) surfactants. If the amount of cetyl alcohol polyoxyethylene ether or surfactant in the composition is insufficient to cause one or more side effects in the individual when the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition) is administered to the individual, the composition is "substantially free of cetyl alcohol polyoxyethylene ether" or "substantially free of surfactants". In some embodiments, the mTOR inhibitor nanoparticle composition (such as sirolimus/albumin nanoparticle composition) contains less than about 20%, 15%, 10%, 7.5%, 5%, 2.5% or 1% of any one of organic solvents or surfactants. In some embodiments, the albumin is human albumin or human serum albumin. In some embodiments, the albumin is recombinant albumin.

The amount of albumin in the mTOR inhibitor nanoparticle composition that can be used with the methods described herein will vary depending on the other components in the composition. In some embodiments, the mTOR inhibitor nanoparticle composition comprises an amount of albumin sufficient to stabilize the mTOR inhibitor (such as a limus drug, such as sirolimus or a derivative or analog thereof) in an aqueous suspension, for example, in the form of a stable colloidal suspension (such as a stable suspension of nanoparticles). In some embodiments, the albumin is in an amount that reduces the sedimentation rate of the mTOR inhibitor (such as a limus drug, such as sirolimus or a derivative thereof) in an aqueous medium. For compositions containing particles, the amount of albumin also depends on the size and density of the nanoparticles of the mTOR inhibitor.

An mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) is "stable" in an aqueous suspension when it remains suspended in an aqueous medium (e.g., without visible precipitation or sedimentation) for an extended period of time, such as at least about any of 0.1, 0.2, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, 60, or 72 hours. The suspension is generally (but not necessarily) suitable for administration to a subject (e.g., a human). The stability of a suspension is generally (but not necessarily) evaluated at a storage temperature, such as room temperature (e.g., 20-25°C) or refrigerated conditions (e.g., 4°C). For example, if the suspension does not exhibit flocculation or particle agglomeration visible to the naked eye about fifteen minutes after the suspension is prepared or does not exhibit flocculation or particle agglomeration when viewed under an optical microscope at 1000x magnification, it is stable at the storage temperature. Stability can also be evaluated under accelerated testing conditions, such as at temperatures above about 40°C.

In some embodiments, albumin is present in the mTOR inhibitor nanoparticle composition in an amount sufficient to stabilize the mTOR inhibitor (e.g., limus drug, e.g., sirolimus or a derivative or analog thereof) at a certain concentration in an aqueous suspension. For example, the concentration of the mTOR inhibitor (e.g., limus drug, e.g., sirolimus or a derivative or analog thereof) in the composition is about 0.1 mg/ml to about 100 mg/ml, including, for example, about 0.1 mg/ml to about 50 mg/ml, about 0.1 mg/ml to about 20 mg/ml, about 1 mg/ml to about 10 mg/ml, about 2 mg/ml to about 8 mg/ml, about 4 mg/ml to about 6 mg/ml, or about 5 mg/ml. In some embodiments, the concentration of the mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) is at least about any one of 1.3 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, and 50 mg/ml. In some embodiments, the albumin is present in an amount such that the composition is free of or substantially free of a surfactant (such as cetyl alcohol polyoxyethylene ether) in order to avoid the use of a surfactant (such as cetyl alcohol polyoxyethylene ether).

In some embodiments, the mTOR inhibitor nanoparticle composition in liquid form comprises about 0.1% to about 50% (w/v) (e.g., about 0.5% (w/v), about 5% (w/v), about 10% (w/v), about 15% (w/v), about 20% (w/v), about 30% (w/v), about 40% (w/v), or about 50% (w/v)) albumin. In some embodiments, the mTOR inhibitor nanoparticle composition in liquid form comprises about 0.5% to about 5% (w/v) albumin.

In some embodiments, the weight ratio of albumin to mTOR inhibitor (e.g., limus drug, e.g., sirolimus or a derivative or analog thereof) in the mTOR inhibitor nanoparticle composition is such that a sufficient amount of the mTOR inhibitor is bound to or transported by cells. Although the weight ratio of albumin to mTOR inhibitor (such as limus drugs, e.g., sirolimus or a derivative or analog thereof) must be optimized for different albumin and mTOR inhibitor combinations, generally the weight ratio (w/w) of albumin to mTOR inhibitor (such as limus drugs, e.g., sirolimus or a derivative or analog thereof) is about 0.01:1 to about 100:1, about 0.02:1 to about 50:1, about 0.05:1 to about 20:1, about 0.1:1 to about 20:1, about 1:1 to about 18:1, about 2:1 to about 15:1, about 3:1 to about 12:1, about 4:1 to about 11:1, about 5:1 to about 10:1, or about 10:1. In some embodiments, the weight ratio of albumin to mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) is about any of 18: 1 or less, 15: 1 or less, 14: 1 or less, 13: 1 or less, 12: 1 or less, 11: 1 or less, 10: 1 or less, 9: 1 or less, 8: 1 or less, 7: 1 or less, 6: 1 or less, 5: 1 or less, 4: 1 or less, and 3: 1 or less. In some embodiments, the weight ratio of albumin (such as human albumin or human serum albumin) to mTOR inhibitor (such as limus drug, e.g., sirolimus or a derivative or analog thereof) in the composition is any one of: about 1:1 to about 18:1, about 1:1 to about 15:1, about 1:1 to about 12:1, about 1:1 to about 10:1, about 1:1 to about 9:1, about 1:1 to about 8:1, about 1:1 to about 7:1, about 1:1 to about 6:1, about 1:1 to about 5:1, about 1:1 to about 4:1, about 1:1 to about 3:1, about 1:1 to about 2:1, about 1:1 to about 1:1.

In some embodiments, albumin allows administration of the mTOR inhibitor nanoparticle composition to an individual (e.g., a human) without significant side effects. In some embodiments, the albumin (e.g., human serum albumin or human albumin) is in an amount effective to reduce one or more side effects of administering an mTOR inhibitor (e.g., a limus drug, e.g., sirolimus or a derivative or analog thereof) to a human. In some embodiments, the term "reducing" one or more side effects of administering an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) refers to reducing, alleviating, eliminating, or avoiding one or more undesirable effects caused by the mTOR inhibitor, as well as side effects caused by the delivery vehicle used to deliver the mTOR inhibitor (such as a solvent that makes the limus drug suitable for injection). Such side effects include, for example, bone marrow suppression, neurotoxicity, allergy, inflammation, venous irritation, phlebitis, pain, skin irritation, peripheral neuropathy, neutropenic fever, allergic reaction, venous embolism, extravasation, and combinations thereof. However, these side effects are merely exemplary and other side effects or combinations of side effects associated with limus drugs (e.g., limus drugs, such as sirolimus or a derivative or analog thereof) may be reduced.

In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) and an albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 200 nm. In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) and an albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 150 nm. In some embodiments, the mTOR inhibitor nanoparticle compositions that can be used with the methods described herein include nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) and an albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles does not exceed about 150 nm (e.g., about 100 nm). In some embodiments, the mTOR inhibitor nanoparticle compositions that can be used with the methods described herein include nanoparticles comprising sirolimus and human albumin (such as human serum albumin), wherein the average diameter of the nanoparticles does not exceed about 150 nm (e.g., about 100 nm). In some embodiments, the mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising sirolimus and human albumin (such as human serum albumin), wherein the average diameter of the nanoparticles is about 10 nm to about 150 nm. In some embodiments, the mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising sirolimus and human albumin (such as human serum albumin), wherein the average diameter of the nanoparticles is about 40 nm to about 120 nm.

In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (e.g., a limus drug, e.g., sirolimus, or a derivative or analog thereof) and albumin (e.g., human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 200 nm, wherein the weight ratio of albumin to mTOR inhibitor (e.g., sirolimus) in the composition is no more than about 10:1 (e.g., about 10:1 or about 9:1 or about 8:1). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (e.g., a limus drug, e.g., sirolimus, or a derivative or analog thereof) and albumin (e.g., human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor (e.g., sirolimus) in the composition is no more than about 10:1 (e.g., about 10:1 or about 9:1 or about 8:1). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (e.g., a limus drug, e.g., sirolimus or a derivative or analog thereof) and albumin (e.g., human albumin or human serum albumin), wherein the nanoparticles have an average diameter of about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor (e.g., sirolimus) in the composition is no more than about 10:1 (e.g., about 10:1 or about 9:1 or about 8:1). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein include nanoparticles comprising sirolimus and human albumin (e.g., human serum albumin), wherein the average diameter of the nanoparticles is no more than about 150 nm (e.g., about 100 nm), wherein the weight ratio of albumin to mTOR inhibitor in the composition is about 10:1, or about 9:1, or about 8:1. In some embodiments, the average diameter of the nanoparticles is about 10 nm to about 150 nm. In some embodiments, the average diameter of the nanoparticles is about 40 nm to about 120 nm.

In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) conjugated to (e.g., coated with) an albumin (such as human albumin or human serum albumin). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) conjugated to (e.g., coated with) an albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 200 nm. In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) conjugated to (e.g., coated with) an albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 150 nm. In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) conjugated to (e.g., coated with) an albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is about 10 nm to about 150 nm. In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein include nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) conjugated to (e.g., coated with) an albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is about 40 nm to about 120 nm. In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein include nanoparticles comprising sirolimus conjugated to (e.g., coated with) a human albumin (such as human serum albumin), wherein the average diameter of the nanoparticles is no more than about 150 nm (e.g., about 100 nm). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising sirolimus conjugated to (e.g., coated with) human albumin (e.g., human serum albumin), wherein the average diameter of the nanoparticles is from about 10 nm to about 150 nm. In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising sirolimus conjugated to (e.g., coated with) human albumin (e.g., human serum albumin), wherein the average diameter of the nanoparticles is from about 40 nm to about 120 nm.

In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) associated with (e.g., coated with) an albumin (such as human albumin or human serum albumin), wherein the weight ratio of albumin to mTOR inhibitor in the composition does not exceed about 10:1 (e.g., about 10:1 or about 9:1 or about 8:1). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) conjugated to (e.g., coated with) an albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 200 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 10:1 (e.g., about 10:1 or about 9:1 or about 8:1). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) associated with (e.g., coated with) an albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 10:1 (e.g., about 10:1 or about 9:1 or about 8:1). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) conjugated to (e.g., coated with) an albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition does not exceed about 10:1 (e.g., about 10:1 or about 9:1 or about 8:1). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein include nanoparticles comprising sirolimus associated with (e.g., coated with) human albumin (e.g., human serum albumin), wherein the average diameter of the nanoparticles is no more than about 150 nm (e.g., about 100 nm), wherein the weight ratio of albumin to sirolimus in the composition is about 10:1, or about 9:1, or about 8:1. In some embodiments, the average diameter of the nanoparticles is about 10 nm to about 150 nm. In some embodiments, the average diameter of the nanoparticles is about 40 nm to about 120 nm.

In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) stabilized with albumin (such as human albumin or human serum albumin). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) stabilized with albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 200 nm. In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) stabilized with albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 150 nm. In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) stabilized with albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 150 nm (e.g., about 100 nm). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein include nanoparticles comprising sirolimus stabilized by human albumin (e.g., human serum albumin), wherein the average diameter of the nanoparticles is no more than about 150 nm (e.g., about 100 nm). In some embodiments, the average diameter of the nanoparticles is about 10 nm to about 150 nm. In some embodiments, the average diameter of the nanoparticles is about 40 nm to about 120 nm.

In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) stabilized by albumin (such as human albumin or human serum albumin), wherein the weight ratio of albumin to mTOR inhibitor in the composition does not exceed about 10:1 (such as about 10:1 or about 9:1 or about 8:1). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) stabilized by albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 200 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 10:1 (such as about 10:1 or about 9:1 or about 8:1). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) stabilized by albumin (such as human albumin or human serum albumin), wherein the average diameter of the nanoparticles is no more than about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition is no more than about 10:1 (such as about 10:1 or about 9:1 or about 8:1). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein comprise nanoparticles comprising an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) stabilized by albumin (such as human albumin or human serum albumin), wherein the nanoparticles have an average diameter of about 150 nm, wherein the weight ratio of albumin to mTOR inhibitor in the composition does not exceed about 10:1 (such as about 10:1 or about 9:1 or about 8:1). In some embodiments, mTOR inhibitor nanoparticle compositions useful with the methods described herein include nanoparticles comprising sirolimus stabilized by human albumin (e.g., human serum albumin), wherein the average diameter of the nanoparticles is no more than about 150 nm (e.g., about 100 nm), wherein the weight ratio of albumin to sirolimus in the composition is about 10: 1, or about 9: 1, or about 8: 1. In some embodiments, the average diameter of the nanoparticles is about 10 nm to about 150 nm. In some embodiments, the average diameter of the nanoparticles is about 40 nm to about 120 nm.

In some embodiments, the mTOR inhibitor nanoparticle composition that can be used with the methods described herein comprises nanoparticle albumin-bound sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition that can be used with the methods described herein is nanoparticle albumin-bound sirolimus. Nanoparticle albumin-bound sirolimus is a sirolimus formulation stabilized by human albumin USP that can be dispersed in a directly injectable physiological solution. The weight ratio of human albumin to sirolimus is about 8:1 to about 10:1. When dispersed in a suitable aqueous medium (such as 0.9% sodium chloride injection or 5% dextrose injection), nanoparticle albumin-bound sirolimus forms a stable colloidal suspension of sirolimus. The average particle size of the nanoparticles in the colloidal suspension is about 100 nanometers. Since HSA is freely soluble in water, nanoparticle albumin-bound sirolimus can be reconstituted in a wide range of concentrations ranging from dilute (0.1 mg/ml sirolimus or its derivative) to concentrated (20 mg/ml sirolimus or its derivative), including, for example, about 2 mg/ml to about 8 mg/ml, or about 5 mg/ml.

Methods for preparing nanoparticle compositions are known in the art. For example, nanoparticles containing an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) and an albumin (such as human serum albumin or human albumin) can be prepared under high shear conditions (e.g., sonication, high pressure homogenization, or the like). Such methods are disclosed in, for example, U.S. Patent Nos. 5,916,596, 6,506,405, 6,749,868, 6,537,579, 7,820,788, and 8,911,786, and in U.S. Patent Publication Nos. 2007/0082838, 2006/0263434, and PCT Application WO 08/137148.

Briefly, an mTOR inhibitor (such as a limus drug, e.g., sirolimus or a derivative or analog thereof) is dissolved in an organic solvent, and the solution can be added to an albumin solution. The mixture is subjected to high pressure homogenization. The organic solvent can then be removed by evaporation. The resulting dispersion can be additionally lyophilized. Suitable organic solvents include, for example, ketones, esters, ethers, chlorinated solvents, and other solvents known in the art. For example, the organic solvent may be dichloromethane or chloroform/ethanol (e.g., a ratio of 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1). Other components in the mTOR inhibitor nanoparticle composition

In some embodiments, the mTOR inhibitor nanoparticle compositions that can be used with the methods described herein are present in a composition that includes other agents, excipients, or stabilizers. For example, certain negatively charged components can be added to improve stability by increasing the negative zeta potential of the nanoparticles. Such negatively charged components include, but are not limited to, bile salts of cholic acid composed of glycocholic acid, cholic acid, chelic acid, taurocholic acid, chelic deoxycholic acid, taurodeoxycholic acid, cholic acid, ursodeoxycholic acid, dehydrocholic acid, and other cholic acids; phospholipids, including those based on lecithin (egg yolk); The phospholipids include the following phospholipid acyl choline: palmityl oleyl phosphatidyl acyl choline, palmityl linoleyl phosphatidyl acyl choline, stearyl linoleyl phosphatidyl acyl choline, stearyl oleyl phosphatidyl acyl choline, stearyl arachidyl phosphatidyl acyl choline and disalmitoyl phosphatidyl acyl choline. Other phospholipids include L-α-dimyristylphosphatidylcholine (DMPC), dioleylphosphatidylcholine (DOPC), distearylphosphatidylcholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC) and other related compounds. Negatively charged surfactants or emulsifiers are also suitable as additives, such as sodium cholesterol sulfate and its analogs.

In some embodiments, the mTOR inhibitor nanoparticle compositions that can be used with the methods described herein are suitable for administration to humans. In some embodiments, the compositions are suitable for administration to mammals, such as in veterinary situations, domestic pets, and farm animals. There are a variety of suitable formulations of mTOR inhibitor nanoparticle compositions (such as sirolimus/albumin nanoparticle compositions) (see, e.g., U.S. Patent Nos. 5,916,596 and 6,096,331). The following formulations and methods are exemplary only and are by no means limiting. Formulations suitable for oral administration may consist of: (a) liquid solutions, such as an effective amount of the compound dissolved in a diluent, such as water, saline or orange juice; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient in solid or particulate form; (c) suspensions in appropriate liquids; and (d) suitable emulsions. Tablet forms may include one or more of the following: lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, gum arabic, gelatin, colloidal silicon dioxide, cross-linked sodium carboxymethyl cellulose, talc, magnesium stearate, stearic acid and other formulators, colorants, diluents, buffers, wetting agents, preservatives, flavoring agents and pharmacologically compatible formulators. Lozenge forms may contain a flavoring, usually the active ingredient in sucrose and acacia or tragacanth, and tablets comprise the active ingredient in an inert matrix such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like which contain, in addition to the active ingredient, excipients such as those known in the art.

Examples of suitable carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methylhydroxybenzoate and propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The formulation may additionally include lubricants, wetting agents, emulsifiers and suspending agents, preservatives, sweeteners or flavoring agents.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation compatible with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. The formulations may be present in unit-dose or multi-dose sealed containers such as ampoules and vials, and may be stored in freeze-dried (lyophilized) conditions requiring only the addition of a sterile liquid excipient such as water immediately prior to use for injection. Ready-to-use injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type previously described. Injectable formulations are preferred.

In some embodiments, the mTOR inhibitor nanoparticle compositions that can be used with the methods described herein are formulated to have a pH range of about 4.5 to about 9.0, including, for example, a pH range of about 5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. In some embodiments, the pH of the composition is formulated to be no less than about 6, including, for example, no less than about 6.5, 7, or 8 (such as about 8). The composition can also be made isotonic with blood by adding a suitable tonicity regulator, such as glycerol. Albumin-based nanoparticle compositions of sirolimus (rapamycin)

In some embodiments, the mTOR inhibitor nanoparticle composition that can be used with the methods described herein includes (a) nanoparticles including sirolimus and albumin, and (b) a non-nanoparticle portion including sirolimus and albumin. The sirolimus and albumin of the nanoparticle are associated with each other in the nanoparticle. For example, the nanoparticle may include a coating with albumin that surrounds a core comprising sirolimus. In the non-nanoparticle portion of the composition, sirolimus and albumin may or may not be associated with each other (i.e., sirolimus may be in a reversible binding equilibrium with albumin), but are not associated with each other in a manner that forms nanoparticles. That is, the nanoparticle composition may include nanoparticle-bound albumin and nanoparticle-bound sirolimus in the nanoparticle portion of the composition, and include non-nanoparticle albumin and non-nanoparticle sirolimus in the non-nanoparticle portion of the composition. As used herein, "in the nanoparticle" is used synonymously with "in the nanoparticle portion." The albumin of the nanoparticle can be further distinguished from the albumin in the non-nanoparticle portion of the composition; for example, the oligomeric distribution of albumin in the nanoparticle can be different from the oligomeric distribution of albumin in the non-nanoparticle portion of the composition. Oligomer distribution means the percentage of various albumin species compared to the total albumin in the composition. Types of albumin species include albumin monomers, dimers, trimers, oligomers, and polymers. As used herein, "albumin monomer" or "monomeric albumin" refers to an albumin species having one and only one albumin unit; "albumin dimer" or "dimeric albumin" refers to an albumin species having two and only two albumin units; "albumin trimer" or "trimeric albumin" refers to an albumin species having three and only three albumin units; "albumin polymer" refers to an albumin species having a higher molecular weight than albumin monomer and albumin dimer; "albumin oligomer" or "oligomeric albumin" refers to a low molecular weight polymeric albumin species associated with a UV-based size exclusion chromatography peak that is observed between the peaks associated with albumin dimer and higher molecular weight polymeric albumin species.

The albumin of the nanoparticles is conjugated to the sirolimus of the nanoparticles, so that the nanoparticle suspension has a high concentration of sirolimus, which allows the composition to be used as a pharmaceutical composition for the treatment of certain diseases, such as cancer. The manufactured nanoparticles (which can be made, for example, using the methods described herein) can be formulated, filtered or otherwise processed to obtain a pharmaceutical composition, which can be suitable for medical use in human subjects.

Generally, to prepare the sirolimus pharmaceutical compositions described herein, sirolimus is dissolved in an organic solvent. Suitable organic solvents include, for example, ketones, esters, ethers, chlorinated solvents, and other solvents known in the art. For example, the organic solvent can be a mixture of dichloromethane/ethanol, chloroform/ethanol, or chloroform/tert-butyl alcohol (e.g., at a ratio of about 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1, or at a ratio of about 3:7, 5:7, 4:6, 5:5, 6:5, 8:5, 9:5, 9.5:5, 5:3, 7:3, 6:4, or 9.5:0.5). In some embodiments, the organic solvent comprises between about 10% and about 50% by volume of tert-butyl alcohol. In some embodiments, the organic solvent comprises about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by volume of tertiary butanol. In some embodiments, the organic solvent comprises about 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, or 45-50% by volume of tertiary butanol, or any combination of such ranges. In some embodiments, the organic solvent comprises between about 50% and about 90% by volume of chloroform. In some embodiments, the organic solvent comprises about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% by volume of chloroform. In some embodiments, the organic solvent comprises about 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, or 85-90% by volume of chloroform, or any combination of such ranges. In some embodiments, the organic solvent comprises between about 10% and about 50% by volume of tertiary butanol and between about 50% and about 90% by volume of chloroform. In some embodiments, the organic solvent comprises chloroform and tertiary butanol in a volume ratio of about 1:1 to about 1:9, such as about any one of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and 9:1.

Albumin (e.g., recombinant albumin, e.g., NOVOZYME ^™ recombinant albumin or INTRIVIA ^™ recombinant albumin disclosed herein) is dissolved in an aqueous solution (e.g., water) and combined with a sirolimus solution to form a crude emulsion. The mixture is subjected to high pressure homogenization (e.g., using Avestin, APV Gaulin, MICROFLUIDIZER™ (e.g., MICROFLUIDIZER™ Processor M-110EH from Microfluidics), Stansted, or Ultra Turrax homogenizers). The emulsion can be cycled through the high pressure homogenizer for about 2 to about 100 cycles, such as about 5 to about 50 cycles or about 6 to about 20 cycles (e.g., any of about 6, 8, 10, 12, 14, 16, 18, or 20 cycles). The organic solvent may then be removed by evaporation using suitable equipment known for this purpose, including, but not limited to, a rotary evaporator, a falling film evaporator, a wiped film evaporator, a spray dryer, and similar equipment that can be operated in batch mode or in continuous operation. In some embodiments, the evaporator is a wiped film evaporator. The solvent may be removed under reduced pressure, such as at any of about 25 mm Hg, 30 mm Hg, 40 mm Hg, 50 mm Hg, 100 mm Hg, 200 mm Hg, or 300 mm Hg. The amount of time used to remove the solvent under reduced pressure can be adjusted based on the volume of the formulation. For example, for a formulation produced at a 300 mL scale, the solvent can be removed at about 1 mm Hg to about 300 mm Hg (e.g., any one of about 5 mm Hg-100 mm Hg, 10 mm Hg-50 mm Hg, 20 mm Hg-40 mm Hg, or 25 mm Hg) for about 5 minutes to about 60 minutes (e.g., any one of about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 25, or 30 minutes). The resulting dispersion can be additionally lyophilized.

The nanoparticle compositions (such as pharmaceutical compositions) comprising sirolimus and albumin described herein can be in liquid (e.g., as a nanoparticle suspension) or powder form. For example, in some embodiments, the composition is a liquid nanoparticle suspension (e.g., before lyophilization). In some embodiments, the composition is a reconstituted suspension (e.g., in an aqueous solution such as a saline solution). In some embodiments, the composition is dried, such as lyophilized. In some embodiments, the composition is sterile. In some embodiments, the composition is contained in a sealed container, such as a sealed vial (e.g., a glass bottle) or a sealed bag.

Additional details about sirolimus/albumin nanoparticle compositions are provided in PCT/US2020/060070, the contents of which are incorporated herein in their entirety.

An exemplary sirolimus/albumin nanoparticle composition is FYARRO™, also known as sirolimus protein-bound particles (nanoparticle albumin-bound) for injectable suspension. Complete information about the preparation, formulation, dosing, and administration schedule of sirolimus protein-bound particles (nanoparticle albumin-bound) for injectable suspension (i.e., FYARRO™) can be found in the local drug instructions (for the United States, see, e.g., www.accessdata.fda.gov/drugsatfda_docs/label/2021/213312lbl.pdf). KRAS and Exemplary KRAS Inhibitors

KRAS, a member of the RAS family, is a key regulator of signaling pathways responsible for cell proliferation, differentiation, and survival. See, e.g., Cox et al. (2003) Nat Rev Drug Discov. 13(11): 828-851 and Downward J. (2003) Nat Rev Cancer. 3(1): 11-22. KRAS is the most frequently mutated oncogene in human cancers and mutations in KRAS can cause sustained cell proliferation and cancer development. The distribution of KRAS mutations varies in different human cancers. At the allele level, most single nucleotide substitution mutations occur at one of five "hotspot" codons: 12, 13, 61, 117, and 146. Glycine 12 can mutate into at least eight amino acids (A, C, D, F, L, R, S, and V). Glycine 13 can mutate into at least seven amino acids (A, C, F, P, R, S and V). Glutamine 61 can mutate into at least six other amino acids (E, H, K, L, P and R) and a stop codon. Lysine 117 can mutate into at least two other amino acids (N and R). Alanine 146 can mutate into one of at least six amino acids (E, G, P, S, T and V). In some embodiments, the term "KRAS inhibitor" refers to any agent that inhibits the activity of KRAS mutant protein, such as a polypeptide, fusion polypeptide, antibody, peptide, antisense oligonucleotide or small molecule drug. KRAS G12C inhibitor

The KRAS G12C mutation occurs in approximately 13% of NSCLC patients and 1% to 3% of colorectal and other solid tumors. G12C is a single-point mutation that replaces glycine with cysteine at codon 12. See Cox et al. (2003) Nat Rev Drug Discov. 13(11): 828-851; Neumann et al. (2009) Pathol Res Pract. 205: 858-862; and Biernacka et al. (2016) Cancer Genet. 209(5): 195-198. This substitution favors the activated GTP-bound state of KRAS, thereby amplifying the signaling pathway that leads to tumorigenesis (see, e.g., Ryan et al. (2018) Nat Rev Clin Oncol. 15(11): 709-720).

In some embodiments, the term "KRAS G12C inhibitor" refers to any agent that inhibits the activity of KRAS G12C mutant protein, such as a polypeptide, fusion polypeptide, antibody, peptide, antisense oligonucleotide or small molecule drug. In some embodiments, the KRAS G12C inhibitor directly interacts with the KRAS G12C mutant protein to inhibit the activity of the protein. In some embodiments, the KRAS G12C inhibitor is a small molecule drug. Exemplary small molecule KRAS G12C inhibitors that can be used with the methods provided herein include, but are not limited to, for example, sotolacib (also known as AMG 510, LUMAKRAS™, and LUMYKRAS™), adagracib (also known as MRTX849), JAB-21822 (also known as JAB-21000), GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286, LY3537982, RMC-6291, RMC-8839, HBI-2438, or JNJ-74699157. Other exemplary small molecule KRAS G12C inhibitors are described, for example, in Hillig et al. (2019) Proceedings of the National Academy of Sciences of the United States of America 116(7): 2551-2560 and Sun et al. (2012) Angew Chem Int Ed Engl. 51(25): 6140-6143 and in the following: WO 2020/233592, WO 2021/023247, WO 2021/083167, WO 2020/238791, WO 2021/000885, CN 112585129, CN 112552295, CN 112390818, CN 112390796, WO 2019/141250, CN 111592528, WO 2020/259432, CN 112300153, CN 112552294, WO 2020/239123, CN 110698378, CN 111377918, CN 111205286, CN 112047939, WO 2020/259513, WO 2021/023154, CN 112430234, WO 2021/063346, WO 2021/058018, CN 112225734, WO 2021/155716, CN 112159405, CN 112778302, CN 112830928, WO 2020/1027943, CN 112574199、WO 2021/037018、CN 110172089、WO 2020/156285、CN 111499634、WO 2020/177629、WO 2020/216190、WO 2020/221239、WO 2020/233592、WO 2020/238791、WO 2020/239077、WO 2020/239123、CN 112047933、CN 112047937、CN 112047948、WO 2020/259513、WO 2020/259573、WO 2020/259432、WO 2021/000885, CN 112174950, CN 112300153, WO 2021/023154, WO 2021/023247, CN 112390818, WO 2021/027943, WO 2021/027911, WO 2021/031952, CN 112390796, WO 2021/037018, CN 112442029, WO 2021/043322, WO 2021/052499, CN 112538084, CN 112552295, WO 2021/058018, WO 2021/063346, WO 2021/068898, WO 2021/078285, CN 112225734, CN 112707905, CN 112745335, WO 2021/083167, CN 112778284, WO 2021/088458, CN 112851663, WO 2021/093758, WO 2021/098859, CN 112830928, CN 111377918, WO 2021/104431, WO 2021/109737, WO 2021/113595, CN 112920183, WO 2021/121371, WO 2021/121367, CN 113004269, WO 2021/129824, WO 2021/129820, CN 113061132, WO 2021/139678, WO 2021/139748, CN 111205286, WO 2021/143693, CN 113135924, WO 2021/147965, WO 2021/155716, WO 2021/168193, WO 2021/169990, WO 2021/169963, CN 113321654, WO 2021/175199, WO 2021/180181, WO 2021/185233, WO 2021/190467, WO 2021/197499, CN 112574199 and CN 112300269, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, a method of treating cancer in an individual (e.g., a human) is provided, comprising administering to the individual (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin ("mTOR inhibitor nanoparticle composition"); and (b) an effective amount of a KRAS G12C inhibitor, wherein the KRAS G12C inhibitor is sotolacib (also known as AMG 510, LUMAKRAS™ and LUMYKRAS™), adagracib (also known as MRTX849), JAB-21822 (also known as JAB-21000), GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286, LY3537982, RMC-6291, RMC-8839, HBI-2438, and JNJ-74699157. In some embodiments, the cancer comprises one or more cancer cells expressing KRAS G12C mutant protein. Additionally or alternatively, in some embodiments, the cancer comprises one or more cells having at least one mTOR activating aberration. In some embodiments, the cancer is a solid tumor, lung cancer, bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, or a tumor of unknown origin. In some embodiments, the cancer or tumor (such as any of the aforementioned cancers or tumors) is advanced, unresectable and/or metastatic. In some embodiments, the cancer is a solid tumor (e.g., an advanced, unresectable and/or metastatic solid tumor), lung cancer (e.g., advanced, unresectable and/or metastatic lung cancer), or bladder cancer (e.g., advanced, unresectable and/or metastatic bladder cancer). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), such as advanced, unresectable and/or metastatic NSCLC.

In some embodiments, the KRAS G12C inhibitor is sotolacib, which, as mentioned above, is also known as AMG 510, LUMAKRAS™, and LUMYKRAS™. AMG 510 is currently under development at Amgen/Beiji Shenzhou. Sotolacib can exist in either of two hysteresis isomeric forms and one form is more active than the other (see, e.g., https://cen.acs.org/pharmaceuticals/drug(dash)discovery/Amgen(dash)unveils(dash)KRas(dash)inhibitor(dash)human/97/i14). Sotolacib selectively forms an irreversible covalent bond with the sulfur atom in the cysteine residue that is present in the G12C mutant form of the KRAS protein and not present in the wild-type form. Covalent binding of sotolacib to KRAS G12C locks the protein in its inactive GDP-bound conformation, thereby inhibiting KRAS-dependent signaling. Sotolacib has an empirical formula of C30HF2N6O3 and a molecular weight of 560.606 g/mol. Sotolacib is chemically described as 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-(1M)-1-[4-methyl-2-(propan-2-yl)pyridin-3-yl]-4-[(2S)-2-methyl-4-(prop-2-enyl)piperazin-1-yl]pyrido[2,3-d]pyrimidin-2(1H)-one and has the following chemical structure: The CAS registration number of Sotolacib is 2252403-56-6. The efficacy of Sotolacib has been demonstrated in a subset of patients participating in a single-arm, open-label, multicenter trial (NCT03600883) and is currently being studied in further clinical trials. Complete information about the preparation, formulation, dosing, and administration schedule of Sotolacib can be found in the local drug instructions (for the United States, see, for example, www.accessdata.fda.gov/drugsatfda_docs/label/2021/214665s000lbl.pdf). Additional details about the structure and synthesis of Sotolacib are provided in WO 2018/217651, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the KRAS G12C inhibitor is adagracib (also known as MRTX849). Adaglasib is currently under development at Mirati/Zai Lab. Similar to sotolacib, adagracib selectively forms an irreversible covalent bond with the sulfur atom in the cysteine residue that is present in the G12C mutant form of the KRAS protein and absent in the wild-type form. Similar to sotolacib, the covalent binding of adagracib to KRAS G12C locks the protein in its inactive GDP-bound conformation, thereby inhibiting KRAS-dependent signaling. Adaglasib has an empirical formula of C32H35ClFN7O2 and a molecular weight of 604.13 g/mol. Adaglasib is chemically described as 2-[(2S)-4-[7-(8-chloronaphthalen-1-yl)-2-[[(2S)-1-methylpyrrolidin-2-yl]methoxy]-6,8-dihydro-5H-pyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroprop-2-enyl)piperazin-2-yl]acetonitrile and has the following chemical structure: The CAS registration number of MRTX849 is 2326521-71-3. Adalasib is currently being evaluated in several clinical trials, including NCT04613596, NCT04685135, NCT03785249, NCT04330664, and others. Additional details on the structure and synthesis of adagrasib are provided in Fell et al. (2020) J. Med. Chem. 63, 6679-6693 and WO 2017/201161, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the KRAS G12C inhibitor is JAB-21822. JAB-21822 was developed by Jacobs Pharmaceuticals Group Ltd. (see, e.g., en.jacobiopharma.com/blank3.html?introId=23 and www.jacobiopharma.com/news/207.html). JAB-21822 is currently being evaluated in several clinical trials, including NCT05009329 and NCT05002270. Additional details on the structure and synthesis of JAB-21822 are provided in WO 2021/057832, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the KRAS G12C inhibitor is GDC-6036. GDC-6036 is developed by Genentech (see, e.g., www.genentechoncology.com/pipeline-molecules/kras-g12c.html) and is currently being evaluated in clinical trial NCT04449874. Additional details on the structure and synthesis of GDC-6036 are provided in WO 2020/097537, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the KRAS G12C inhibitor is JDQ443. JDQ443 is a KRAS G12C inhibitor developed by Novartis. The structure of JDQ443 is: JDQ443 is currently being evaluated in clinical trial NCT04699188. Additional details on the synthesis of JDQ443 are provided in WO 2021/124222, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the KRAS G12C inhibitor is D-1553. D-1553 was developed by Inventis Biotech Co., Ltd. (see, e.g., www.inventisbio.com/%e4%b8%b4%e5%ba%8a%e8%af%95%e9%aa%8c/) and is currently being evaluated in clinical trial NCT04585035 in collaboration with Merck. Additional details on the structure and synthesis of D-1553 are provided in WO 2020/233592, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the KRAS G12C inhibitor is GH35. GH35 was developed by Suzhou Genhouse Pharmaceutical Co., Ltd. (see, e.g., www.genhousebio.com/en/product/index.html) and evaluated in clinical trial NCT05010694. Additional details on the structure and synthesis of GH35 are provided in WO 2020/177653, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the KRAS G12C inhibitor is GFH925. GFH925 was developed by Genfleet Pharmaceuticals (Zhejiang) (see, e.g., www.genfleet.com/en/science) and is being evaluated in clinical trial NCT05005234. Additional details on the structure and synthesis of GFH925 are provided in WO 2020/177629, WO 2020/221239, and WO 2021/031952, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the KRAS G12C inhibitor is BPI-421286. BPI-421286 was developed by Betta Pharmaceuticals Co., Ltd. (see, e.g., www.bettapharma.com/News/show/id/2380), and clinical trial applications (CXHL2100046 and CXHL2100047) for evaluating BPI-421286 have been accepted by the State Food and Drug Administration of the People's Republic of China. Additional details on the structure and synthesis of BPI-421286 are provided in CN 112390796, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the KRAS G12C inhibitor is LY3537982. LY3537982 is developed by Eli Lilly and Loxo Oncology (see, e.g., www.lillyloxooncologypipeline.com/molecule/kras-g12c-inhibitor/) and is being studied in clinical trial NCT04956640. Additional details on the structure and synthesis of LY3537982 are provided in WO 2021/118877, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the KRAS G12C inhibitor is RMC-6291. RMC-6291 is currently under development at Revolution Medicines (see, e.g., www.revmed.com/pipeline) and is being studied in clinical trial NCT05462717. In some embodiments, the KRAS G12C inhibitor is RMC-8839, which is also under development at Revolution medicines.

In some embodiments, the KRAS G12C inhibitor is HBI-2438, which is currently under development by Shanghai Asia Biosciences International, Ltd. HBI-2438 is being studied in clinical trial NCT05485974.

In some embodiments, the KRAS G12C inhibitor is JNJ-74699157 (also known as ARS-3248), which is under development by Johnson & Johnson and Wellspring Bioscience. JNJ-74699157 is being studied in clinical trial NCT04006301.

As discussed above, in some embodiments, the KRAS G12C inhibitor and the mTOR inhibitor nanoparticle composition (such as sirolimus/albumin nanoparticle composition) are administered in a single unit dose. In some embodiments, the KRAS G12C inhibitor and the mTOR inhibitor nanoparticle composition (such as sirolimus/albumin nanoparticle composition) are administered in separate dosage forms. KRAS G12D inhibitor

The incidence of KRAS G12D mutation is high in pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), lung cancer, and colorectal cancer, and is strongly correlated with poor prognosis. G12D substitution also favors the activated GTP-bound state of KRAS, thereby expanding the signaling pathway that leads to tumor formation (see, e.g., Lee et al. (2021) Chem. Sci. 12, 12827-12837).

In some embodiments, the term "KRAS G12D inhibitor" refers to any agent that inhibits the activity of a KRAS G12D mutant protein, such as a polypeptide, a fusion polypeptide, an antibody, a peptide, an antisense oligonucleotide, or a small molecule drug. In some embodiments, a KRAS G12D inhibitor directly interacts with a KRAS G12D mutant protein to inhibit the activity of the protein. In some embodiments, a KRAS G12D inhibitor is a small molecule drug. Exemplary KRAS G12D inhibitors that can be used with the methods provided herein include, but are not limited to, MRTX1133 (Mirati Therapeutics) and RMC-6236 (Revolution Medicines).

In some embodiments, the KRAS ^G12D inhibitor is MRTX1133 (CAS Reg. No. 2621928-55-8), which is under development at Mirati Therapeutics (see, e.g., www.mirati.com/science/programs/kras-inhibitors/kras-g12d-inhibitor/). The structure of MRTX1133 is: Additional details about MRTX1133 are provided in Wang et al. (2022) J Med Chem 65(4): 3123-3133, KRAS G12D inhibitor MRTX1133 illustrates KRAS-mediated tumorigenesis, Nat Med (2022) (doi.org/10.1038/s41591-022-02008-6) and Hallin, Bowcut, Calinisan et al., Antitumor efficacy of a potent and selective non-covalent KRAS G12D inhibitor, Nat Med (2022) (doi.org/10.1038/s41591-022-02007-7), the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the KRAS G12D inhibitor is RMC-9805, which is under development at Revolution Medicines (see, e.g., www.revmed.com/pipeline/rason-inhibitors).

As discussed above, in some embodiments, the KRAS G12D inhibitor and the mTOR inhibitor nanoparticle composition (such as sirolimus/albumin nanoparticle composition) are administered in a single unit dose. In some embodiments, the KRAS G12D inhibitor and the mTOR inhibitor nanoparticle composition (such as sirolimus/albumin nanoparticle composition) are administered in separate dosage forms. KRAS G12V inhibitor

The incidence of KRAS G12V mutation is higher in pancreatic cancer (e.g., pancreatic duct adenocarcinoma), lung cancer, and colorectal cancer. In some embodiments, the term "KRAS G12V inhibitor" refers to any agent that inhibits the activity of KRAS G12V mutant protein, such as a polypeptide, a fusion polypeptide, an antibody, a peptide, an antisense oligonucleotide, or a small molecule drug. In some embodiments, the KRAS G12D inhibitor directly interacts with the KRAS G12V mutant protein to inhibit the activity of the protein. In some embodiments, the KRAS G12V inhibitor is a small molecule drug. Exemplary KRAS G12V inhibitors that can be used with the methods provided herein include, but are not limited to, JAB-23000 (Jacco Pharmaceuticals). JAB-23000 is under development by Jacco Pharmaceuticals. Additional details about JAB-23000 are provided in Reck et al. (2001) Annals of Oncology . 32(9): 1101-1110 and entzz.jacobiopharma.com/upload/202108/20210831215444372.pdf, the contents of which are incorporated herein by reference in their entirety.

As discussed above, in some embodiments, the KRAS G12V inhibitor and the mTOR inhibitor nanoparticle composition (such as sirolimus/albumin nanoparticle composition) are administered in a single unit dose. In some embodiments, the KRAS G12V inhibitor and the mTOR inhibitor nanoparticle composition (such as sirolimus/albumin nanoparticle composition) are administered in separate dosage forms. Selecting an individual for treatment according to the methods herein mTOR activating aberrations

As discussed above, in some embodiments, the methods herein include (e.g., further include) selecting an individual for treatment based on the presence of one or more cancer cells having at least one mTOR activating aberration in a sample of the individual prior to administering an mTOR inhibitor nanoparticle composition and a KRAS G12C inhibitor. In some embodiments, "mTOR activating aberration" refers to a genetic aberration, an abnormal expression amount and/or an abnormal activity level of one or more mTOR-related genes that can cause overactivation of the mTOR signaling pathway. In some embodiments, "overactivation" refers to an increase in the activity level of a molecule (such as a protein or protein complex) or a signaling pathway (such as an mTOR signaling pathway) to a level above a reference activity level or range, such as at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, 100%, 200%, 500% or more above the reference activity level or the median of the reference activity range. In some embodiments, the reference activity level is a clinically acceptable normal activity level in a standardized test, or the activity level in a healthy individual (or a tissue or cell isolated from an individual) that does not have an mTOR activation aberration. In some embodiments, at least one mTOR activating aberration is an aberration associated with one or more mTOR-related genes (e.g., AKT1, FLT-3, MTOR, PIK3CA, PIK3CG, TSC1, TSC2, RHEB, STK11, NF1, NF2, TP53, FGFR4, BAP1, KRAS, NRAS, NRF2, KEAP1, and PTEN), including deviations from a reference sequence (i.e., genetic aberrations), abnormal expression and/or abnormal activity levels of one or more proteins encoded by mTOR-related genes. In some embodiments, at least one mTOR activating aberration is an aberration associated with TSC1 and/or TSC2. In some embodiments, at least one mTOR activating aberration comprises STK11 and/or TP53 aberrations. Exemplary STK11 aberrations include STK11 E317, SKT11 null, and any mutation in Figure 1 of Oncologist. 2020 Sep; 25(9): 733-737, which is incorporated by reference in its entirety. Exemplary TP53 aberrations include G262 (e.g., G262V), C176 (e.g., C176F), F113 (e.g., F113C), and any mutation described in Figure 1 of Front Oncol. 2020; 10: 593383, which is incorporated by reference in its entirety. Such exemplary TP53 mutations include loss-of-function mutations (e.g., R175H, G245S, R248Q, R248W, S241F, R249S, R273C, R273H, C275Y, R280K), partial-function and/or temperature-sensitive mutations (e.g., A161T, R181L, R202S, Y220H, S215C, D228V, V272L, R282W), mutants or hyper-transcriptional activation Mutations (e.g., T123A, G199H, S240N, S288K, R337H, G360V), specific changes (K120R, S121F, V122A, T125R, G279E), dominant mutations (e.g., R175H, G245S, R248Q, R248W, S241F, R249S, R273C, R273H, C275Y, R280K), and D281G, R282W. In some embodiments, the mTOR inhibitor aberrations comprise PTEN aberrations (e.g., PTEN null) and/or PIK3CA aberrations (e.g., PIK3CA mutants). In some embodiments, the individual further comprises ATM (e.g., Q2800fs), CDKN2A (e.g., CDKN2A null), and/or UGT2B17 aberrations (e.g., UGT2B17 null).

In some embodiments, at least one mTOR activating aberration is a genetic aberration. Genetic aberrations of one or more mTOR-related genes may include changes in nucleic acid (such as DNA and RNA) or protein sequences (i.e., mutations) or epigenetic characteristics associated with the mTOR-related genes described herein, including but not limited to coding, non-coding, regulatory, enhancer, silencer, promoter, intron, exon, and non-translational regions of mTOR-related genes.

In some embodiments, the genetic aberration may be a germline mutation (including chromosome rearrangement) or a somatic cell mutation (including chromosome rearrangement). In some embodiments, the genetic aberration is present in all tissues of the individual, including normal tissues and cancer tissues. In some embodiments, the genetic aberration is present only in cancer tissues of the individual. In some embodiments, the genetic aberration is present only in a portion of cancer tissues.

In some embodiments, the mTOR activation aberration comprises a mutation of an mTOR-related gene described herein, including but not limited to deletion, frame shift, insertion, insertion/deletion, missense mutation, nonsense mutation, point mutation, single nucleotide variation (SNV), silent mutation, splice site mutation, splice variant, and translocation. In some embodiments, the mutation may be a loss-of-function mutation of a negative regulator of the mTOR signaling pathway or a gain-of-function mutation of a positive regulator of the mTOR signaling pathway. In some embodiments, the mutation of an mTOR-related gene is a mutation of TSC1 or TSC2.

In some embodiments, the gene aberration comprises a change in the number of copies of the mTOR-related genes described herein. Typically, there are two copies of each mTOR-related gene in each genome. In some embodiments, the number of copies of mTOR-related genes is increased by gene aberration, thereby generating at least about 3, 4, 5, 6, 7, 8 or more copies of mTOR-related genes in the genome. In some embodiments, the gene aberration of mTOR-related genes causes the loss of one or two copies of mTOR-related genes in the genome. In some embodiments, the number of copies of mTOR-related genes changes to a heterozygous deletion of mTOR-related genes. In some embodiments, the number of copies of mTOR-related genes changes to a deletion of mTOR-related genes. In some embodiments, the copy number change of the mTOR-related gene is caused by the structural rearrangement of the genome, including the deletion, duplication, inversion and translocation of the chromosome or its fragment. In some embodiments, the copy number change of the mTOR-related gene is the copy number change of TSC1 or TSC2.

In some embodiments, the genetic aberration comprises an abnormal epigenetic characteristic associated with an mTOR-related gene described herein, including but not limited to DNA methylation, hydroxymethylation, abnormal histone binding, chromosomal remodeling, and the like. In some embodiments, the promoter of an mTOR-related gene is hypermethylated in an individual, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, compared to a control level (e.g., a clinically acceptable normal level in a standardized test). In some embodiments, the abnormal epigenetic characteristic associated with an mTOR-related gene is associated with TSC1 or TSC2.

One or more genetic aberrations of one or more mTOR-related genes (e.g., TSC1 or TSC2) described herein can be assessed based on a sample, such as a sample from an individual and/or a reference sample. In some embodiments, the sample is a tissue sample or nucleic acid extracted from a tissue sample (e.g., a tumor tissue sample). In some embodiments, the sample is a cell sample or nucleic acid extracted from a cell sample (e.g., a CTC sample). In some embodiments, the sample is a tumor biopsy. In some embodiments, the sample is a tumor sample or nucleic acid extracted from a tumor sample. In some embodiments, the sample is a biopsy sample or nucleic acid extracted from a biopsy sample. In some embodiments, the sample is a formaldehyde-fixed paraffin-embedded (FFPE) sample or nucleic acid extracted from a FFPE sample. In some embodiments, the sample is a blood sample. In some embodiments, free DNA is separated from a blood sample. In some embodiments, the biological sample is a plasma sample or nucleic acid extracted from a plasma sample.

Genetic aberrations of mTOR-related genes can be determined by any method known in the art. See, for example, Dickson et al., Int. J. Cancer, 2013, 132(7): 1711-1717; Wagle N. Cancer Discovery, 2014, 4: 546-553; and the Cancer Genome Atlas Research Network, Nature 2013, 499: 43-49. Exemplary methods include, but are not limited to, genome DNA sequencing, bisulfite sequencing, or other DNA sequencing-based methods using Sanger sequencing or next-generation sequencing platforms; polymerase chain reaction analysis; in situ hybridization analysis; and DNA microarrays. Epigenetic features (e.g., DNA methylation, histone binding, or chromosomal modification) of one or more mTOR-related genes from a sample isolated from an individual can be compared to epigenetic features of one or more mTOR-related genes from a control sample. Nucleic acid molecules extracted from the sample can be sequenced or analyzed for the presence of mTOR-activating gene aberrations relative to reference sequences, such as wild-type sequences of AKT1, FLT-3, MTOR, PIK3CA, PIK3CG, TSC1, TSC2, RHEB, STK11, NF1, NF2, TP53, FGFR4, BAP1, KRAS, NRAS, NRF2, KEAP1, and PTEN.

In some embodiments, free DNA sequencing methods are used to assess genetic aberrations in mTOR-related genes. In some embodiments, next-generation sequencing is used to assess genetic aberrations in mTOR-related genes. In some embodiments, next-generation sequencing is used to assess genetic aberrations in mTOR-related genes isolated from blood samples. In some embodiments, exome sequencing is used to assess genetic aberrations in mTOR-related genes. In some embodiments, fluorescent in situ hybridization analysis is used to assess genetic aberrations in mTOR-related genes. In some embodiments, genetic aberrations in mTOR-related genes are assessed before initiating the treatment methods described herein. In some embodiments, genetic aberrations in mTOR-related genes are assessed after initiating the treatment methods described herein. In some embodiments, genetic aberrations in mTOR-related genes are assessed before and after initiating the treatment methods described herein. Aberration of gene mTOR activation in TSC1 and TSC2

In some embodiments, the gene mTOR activation aberration comprises an in-frame deletion mutation in TSC1 or TSC2. In some embodiments, the in-frame deletion mutation has been reported in the Leiden Open Variant Database ("LOVD", available at, for example, databases.lovd.nl/shared/genes/TSC2). In some embodiments, the in-frame deletion mutation in TSC1 or TSC2 deletes more than one amino acid of a certain size. In some embodiments, the gene mTOR activation aberration comprises a missense mutation in TSC1. In some embodiments, the missense mutation in TSC1 comprises a non-conservative substitution within amino acids 34-224 or exons 4-8 of TSC1. In some embodiments, the gene mTOR activation aberration comprises a missense mutation in TSC2. In some embodiments, the missense mutation in TSC2 comprises a non-conservative substitution and/or has been reported in the LOVD database. In some embodiments, the gene mTOR activation aberration comprises a homozygous deletion mutation. In some embodiments, the homozygous deletion mutation affects one or more exons of TSC1 or TSC2.

Details of methods for evaluating whether one or more mutations in TSC1 or TSC2 are mTOR activating aberrations (e.g., "pathogenic mutations") are provided in PCT/US2020/060070, the contents of which are incorporated herein in their entirety. TSC2

TSC2 is also known as tuberin, tuberous sclerosis complex 2 protein, protein phosphatase 1 regulatory subunit 160, TSC4, PPP1R160, and LAM. TSC2 protein acts as part of a complex with TSC1 by negatively regulating mTORC1 signaling. In some embodiments, the nucleic acid sequence of the wild-type TSC2 gene is identified by GenBank accession number NC_000016.10 on the positive strand of chromosome 16 as nucleotide 2047936 to nucleotide 2088712 according to the GRCh38.p2 set of human genomes. The wild-type TSC2 gene comprises 42 exons. Mutations in the TSC2 gene may occur in any one or any combination of the 42 exons, or in any intron or non-coding region of the TSC2 gene.

In some embodiments, the amino acid sequence of the wild-type TSC2 protein is identified by GenBank Accession No. NP_000539.2. In some embodiments, the amino acid sequence of the wild-type TSC2 protein is identified by GenBank Accession No. NP_001070651.1. In some embodiments, the amino acid sequence of the wild-type TSC2 protein is identified by GenBank Accession No. NP_001107854.1.

In some embodiments, the nucleic acid sequence of the cDNA encoding the wild-type TSC2 protein is identified by GenBank Accession No. NM_000548.3. In some embodiments, the nucleic acid sequence of the cDNA encoding the wild-type TSC2 protein is identified by GenBank Accession No. NM_001077183.1. In some embodiments, the nucleic acid sequence of the cDNA encoding the wild-type TSC2 protein is identified by GenBank Accession No. NM_001114382.1.

In some embodiments, the subject is selected for treatment based on having an mTOR activating aberration at TSC2. In some embodiments, the mTOR activating aberration at TSC2 comprises a mutation (e.g., a non-activating mutation) in TSC2. In some embodiments, the mutation is selected from the group consisting of a splice site mutation, a nonsense mutation, a frame shift mutation, a missense mutation, and a loss or deletion of a gene. In some embodiments, the mTOR activating aberration at TSC2 comprises a single nucleotide variant (SNV). In some embodiments, the SNV comprises a mutation selected from the group consisting of: C1503T, C2743G, C5383T, C3755G, G760T, C3442T, G880A, T707C, A4949G, or a deletion of any one or more of the amino acids at the following positions: 1405-1409, 1960-1970, 4999, 5002, 3521, 5208, 5238-5255.

In some embodiments, the mutation is a double point mutation (i.e., a double allele mutation). In some embodiments, the mutation comprises a triple point mutation or a quadruple point mutation. In some embodiments, the mTOR activating aberration at TSC2 is a loss-of-function mutation. In some embodiments, the mTOR activating aberration at TSC2 comprises a homozygous deletion. In some embodiments, the mTOR activating aberration at TSC2 comprises a copy number variation of TSC2. In some embodiments, the mTOR activating aberration at TSC2 comprises an abnormal expression amount of TSC2. In some embodiments, the mTOR activating aberration at TSC2 comprises an abnormal activity level of a protein encoded by TSC2.

In some embodiments, the subject has a mutation (e.g., an inactivating mutation) in any one or more of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44 according to GenBank Accession No. NM_000548. In some embodiments, the subject has a double allelic mutation (e.g., a double allelic inactivating mutation) in two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44 according to GenBank Accession No. NM_000548. In some embodiments, the subject has an inactivating mutation in any of exons 18, 22, 27, 30, and 42 of TSC2. In some embodiments, the individual has double allelic mutations in any two of TSC2 exons 18, 22, 27, 30, and 42. In some embodiments, the individual has double allelic mutations in TSC2 exons 18 and 30. In some embodiments, the individual has double allelic mutations in TSC2 exons 22 and 27.

In some embodiments, the mutation is not within amino acids 947-989 or exon 26. In some embodiments, the mutation is not within amino acids 1272-1295 or exon 32.

In some embodiments, the mutation comprises a non-conservative substitution.

In some embodiments, the mutation has been reported from the LOVD database.

TSC1 and TSC2 gene mutations are described, e.g., in Rosset et al., Genetics and Molecular Biology, 40, 1, 69-79 (2017), which is incorporated herein in its entirety. In some embodiments, the subject has a contiguous deletion (e.g., a TSC2-PKD1 deletion). See, e.g., Boronat et al., Brain Dev. 36: 801-806. In some embodiments, the subject has a c.5238-5255 deletion in TSC2. See, e.g., Rok et al., Med Sci Monit 11: 230-234. In some embodiments, the subject has a proximal region mutation (e.g., in any one of exons 1-22) and/or a distal region mutation (e.g., in any one of exons 23-41). See, e.g., van Eeghena et al., Epilepsy Res 103: 83-87. TSC1

TSC1 is also known as hamartoma protein, tuberous sclerosis 1 protein, TSC, KIAA0243, and LAM. TSC1 protein acts as part of a complex with TSC2 by negatively regulating mTORC1 signaling. In some embodiments, the nucleic acid sequence of the wild-type TSC1 gene is identified by GenBank accession number NC_000009.12 on the antisense of chromosome 9 as nucleotides 132891348 to nucleotides 132945370 according to the GRCh38.p2 set of human genomes. The wild-type TSC1 gene comprises 25 exons. Mutations in the TSC1 gene may occur in any one or any combination of the 25 exons, or in any intron or non-coding region of the TSC1 gene.

In some embodiments, the amino acid sequence of the wild-type TSC1 protein is identified by GenBank Accession No. NP_000359.1. In some embodiments, the amino acid sequence of the wild-type TSC1 protein is identified by GenBank Accession No. NP_001155898.1. In some embodiments, the amino acid sequence of the wild-type TSC1 protein is identified by GenBank Accession No. NP_001155899.1.

In some embodiments, the nucleic acid sequence of the cDNA encoding the wild-type TSC1 protein is identified by GenBank Accession No. NM_000368.4. In some embodiments, the nucleic acid sequence of the cDNA encoding the wild-type TSC1 protein is identified by GenBank Accession No. NM_001162426.1. In some embodiments, the nucleic acid sequence of the cDNA encoding the wild-type TSC1 protein is identified by GenBank Accession No. NM_001162427.1.

In some embodiments, the subject is selected for treatment based on having an mTOR activating aberration at TSC1. In some embodiments, the mTOR activating aberration at TSC1 comprises a mutation in TSC1 (e.g., an inactivating mutation). In some embodiments, the mutation is selected from the group consisting of: a splice site mutation, a nonsense mutation, a frame shift mutation, a missense mutation, and a loss or deletion of a gene. In some embodiments, the mTOR activating aberration at TSC1 comprises a single nucleotide variant (SNV). In some embodiments, the mutation is a double point mutation. In some embodiments, the mTOR activating aberration at TSC1 is a loss-of-function mutation. In some embodiments, the mTOR activating aberration at TSC1 comprises a homotypic combination deletion. In some embodiments, the mTOR activating aberration at TSC1 comprises a change in the copy number of TSC1. In some embodiments, the mTOR activating aberration at TSC1 comprises an abnormal expression amount of TSC1. In some embodiments, the mTOR activating aberration at TSC1 comprises an abnormal activity level of a protein encoded by TSC1.

In some embodiments, the subject has a mutation (e.g., an inactivating mutation) in any one or more of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 according to GenBank Accession No. NM_000368. In some embodiments, the subject has a double allelic mutation (e.g., a double allelic inactivating mutation) in two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 according to GenBank Accession No. NM_000368. In some embodiments, the mutation is not in exon 23. In some embodiments, the mutation is not in the 3' half of exon 22.

In some embodiments, the mutation comprises a non-conservative substitution.

In some embodiments, the mutation has been reported from the LOVD database.

In some embodiments, the individual has a TSC1 loss or deletion. KRAS mutation

In some embodiments, the methods described herein comprise (e.g., further comprise) administering an mTOR inhibitor nanoparticle composition and a KRAS inhibitor (e.g., a KRAS G12C inhibitor, a KRAS G12A inhibitor, a KRAS G12D inhibitor, a KRAS G12F inhibitor, a KRAS G12L inhibitor, a KRAS G12R inhibitor, a KRAS G12S inhibitor, a KRAS G12V inhibitor, a KRAS G13A inhibitor, a KRAS G13C inhibitor, a KRAS G13D inhibitor, a KRAS G13P inhibitor, a KRAS G13R inhibitor, a KRAS G13S inhibitor, a KRAS G13V inhibitor, a KRAS Q61E inhibitor, a KRAS A KRAS Q61H inhibitor, a KRAS Q61K inhibitor, a KRAS Q61L inhibitor, a KRAS Q61P inhibitor, a KRAS Q61R inhibitor, a KRAS K117N inhibitor, a KRAS K117R inhibitor, a KRAS A146E inhibitor, a KRAS A146G inhibitor, a KRAS A146P inhibitor, a KRAS A146S inhibitor, a KRAS A146T inhibitor, or a KRAS A146V inhibitor) has previously been used to identify KRAS mutant proteins (e.g., KRAS G12C mutant protein, KRAS G12A mutant protein, KRAS G12D mutant protein, KRAS G12F mutant protein, KRAS G12L mutant protein, KRAS G12R mutant protein, KRAS G12S mutant protein, KRAS G12V mutant protein, KRAS G13A mutant protein, KRAS G13C mutant protein, KRAS G13D mutant protein, KRAS G13P mutant protein, KRAS G13R mutant protein, KRAS G13S mutant protein, KRAS G13V mutant protein, KRAS Q61E mutant protein, KRAS Q61H mutant protein, KRAS Q61K mutant protein, KRAS Q61L mutant protein, KRAS Q61P mutant protein, KRAS Q61R mutant protein, KRAS K117N mutant protein, KRAS K117R mutant protein, KRAS A146E mutant protein, KRAS A146G mutant protein, KRAS A146P mutant protein, KRAS In some embodiments, the subject is selected for treatment by detecting the presence of one or more cancer cells expressing KRAS A146S mutant protein, KRAS A146T mutant protein, or KRAS A146V mutant protein. In some embodiments, the cancer cells expressing KRAS G12C mutant protein comprise the KRAS G12C mutation.

KRAS mutations can be assessed based on samples, such as samples from an individual and/or reference samples. In some embodiments, the sample is a tissue sample or nucleic acid extracted from a tissue sample (e.g., a tumor tissue sample). In some embodiments, the sample is a cell sample or nucleic acid extracted from a cell sample (e.g., a CTC sample). In some embodiments, the sample is a tumor biopsy. In some embodiments, the sample is a tumor sample or nucleic acid extracted from a tumor sample. In some embodiments, the sample is a biopsy sample or nucleic acid extracted from a biopsy sample. In some embodiments, the sample is a formaldehyde-fixed paraffin-embedded (FFPE) sample or nucleic acid extracted from a FFPE sample. In some embodiments, the sample is a blood sample. In some embodiments, free DNA is isolated from a blood sample. In some embodiments, the biological sample is a plasma sample or nucleic acid extracted from a plasma sample.

In some embodiments, KRAS mutations are assessed (e.g., detected) by nucleic acid sequencing (biteoxy and pyrophosphate sequencing), PCR (including allele-specific PCR and amplification resistance mutation system (ARMS) quantitative PCR and digital PCR), single strand conformation polymorphism analysis, melting curve analysis, probe hybridization methods (including the use of nucleic acid and peptide nucleic acid probes). KRAS mutations (e.g., KRAS G12C mutations) can also be assessed (e.g., detected) using commercially available kits, such as THERASCREEN® assay (DxS, Manchester, UK), PYROMARK® KRAS assay (Qiagen, Valencia, CA, USA), SIGNATURE® KRAS / BRAF assay (Asuragen, Austin, TX, USA), and others. Such methods are described, for example, in Matsunaga et al. (2016) Oncol Lett . 12(1): 150-156; Brychta et al. (2016) Clinical Chemistry , Vol. 62, No. 11: 1482-1491; Nicolazzo et al. (2021) Diagnostics ( Basel ) . 11(12): 2196; and Anderson (2011) Expert Rev Mol Diagn . 11(6): 635-642, and references cited therein. Exemplary platforms for screening KRAS G12C mutations in patient samples include polymerase chain reaction (PCR) for tumor tissue (e.g., Qiagen therascreen KRAS RGQ PCR kit) and next generation sequencing (NGS) for ctDNA (e.g., Resolution Bioscience). Other selection criteria

In some embodiments, the individual treated according to the methods described herein has a malignant solid tumor confirmed by histology, and has a KRAS mutation (e.g., KRAS G12C mutation) in tumor tissue or plasma ctDNA. In some embodiments, the solid tumor is an advanced (e.g., in stage III or stage IV or terminal), unresectable and/or metastatic solid tumor. In some embodiments, the diameter of the tumor is at least about 0.5, 1, 1.25, 1.5, 1.75, or 2 centimeters. In some embodiments, the individual treated according to the methods described herein has a NSCLC confirmed by histology, and has a KRAS mutation (e.g., KRAS G12C mutation) in tumor tissue or plasma ctDNA. In some embodiments, the NSCLC is advanced, unresectable and/or metastatic NSCLC. In some embodiments, the individual is not a candidate for definitive therapy. In some embodiments, there are no available treatments with curative intent for malignant solid tumors and/or NSCLC. In some embodiments, the individual has received prior therapy with platinum compounds and/or checkpoint inhibitors (with any therapeutic intent). In some embodiments, the individual has not received prior therapy with a KRAS inhibitor (e.g., a KRAS G12C inhibitor). In some embodiments, the individual has received prior therapy with a KRAS inhibitor (e.g., a KRAS G12C inhibitor). In some embodiments, the subject has measurable disease according to RECIST 1.1 (see Eisenhauer et al. (2009) Eur J. Cancer 45: 228-247). In some embodiments, the subject is 18 years of age or older. In some embodiments, the subject has a life expectancy of at least 3 months. In some embodiments in which the subject received recent prior systemic therapy or radiation therapy, the recent prior systemic therapy (e.g., chemotherapy, immunotherapy, or investigational agent) and radiation therapy were interrupted for at least 2 weeks prior to the first dose of the mTOR inhibitor nanoparticle composition and/or KRAS inhibitor (e.g., KRAS G12C inhibitor). In some embodiments where the subject has experienced adverse effects from a prior therapy, the subject has recovered from adverse effects of the prior therapy to ≤ Grade 1 (excluding alopecia, peripheral neuropathy, and parameters substituted by other eligibility criteria, such as hematological parameters) at the time of enrollment. In some embodiments, the subject has an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 (see, e.g., www.ecog-acrin.org/resources/ecog-performance-status). In some embodiments, the individual has adequate organ function (including one or more of the following: for example, absolute neutrophil count ≥ 1,500/mm ³ (≥ 1.5 × 10 ⁹ /L); platelet count ≥ 100,000/mm ³ (≥ 100 × 10 ⁹ /L); hemoglobin ≥ 9 g/dL without transfusion for at least 2 weeks; total bilirubin ≤ 1.5 × upper limit of normal (ULN) (≤ 3 × ULN if associated with liver metastasis or Gilbert's disease); aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 3.0 × ULN (≤ 5 × ULN if associated with liver metastasis); creatinine clearance ≥ 50 mL/min or glomerular filtration rate ≥ 50 mL/min/1.73 m ² calculated using a validated prediction equation (eg, Cockcroft-Gault, Modification of Diet in Nephropathy (MDRD), or 24-hour urine output CrCl).

In some embodiments, the individual does not have active brain metastases or carcinomatous meningitis. In some embodiments, the individual has brain metastases and (a) the brain metastases are adequately treated, (b) the individual is neurostable, and (c) if the individual is receiving steroid therapy, the steroid dose is ≤ 10 mg of prazol (or equivalent) per day. In some embodiments, the individual has no history of significant hemoptysis or bleeding within 4 weeks of the first dose of the mTOR inhibitor nanoparticle composition and/or KRAS inhibitor (e.g., KRAS G12C inhibitor). In some embodiments, the individual has not undergone major surgery within 4 weeks of the first dose of treatment with the mTOR inhibitor nanoparticle composition and KRAS inhibitor. In some embodiments, the subject has no history of intestinal disease, inflammatory bowel disease, major gastric surgery, or other gastrointestinal pathology (e.g., uncontrolled nausea, vomiting, malabsorption syndrome). In some embodiments, the subject does not have (or has no history of) any of the following cardiac abnormalities: (a) unstable angina or myocardial infarction; (b) congestive heart failure ≥ New York Heart Association class 3; (c) medical or family history of prolonged corrected QT (QTc) > 480 milliseconds on ECG during the screening period or congenital long QT syndrome; (d) symptomatic or uncontrolled atrial flutter or other clinically significant arrhythmias. In some embodiments, the subject has no history of stroke or transient ischemic episode, for example, within the previous 6 months. In some embodiments, the subject does not have an ongoing need for medications with a known risk of torsades de pointes or a narrow treatment index CYP3A substrate; strong inhibitors or inducers of CYP3A and/or P-gp; strong inhibitors of breast cancer resistance protein (BCRP); and proton pump inhibitors that cannot be switched to alternative therapy prior to the first dose of treatment with the mTOR inhibitor nanoparticle composition and/or KRAS inhibitor (e.g., KRAS G12C inhibitor). In some embodiments, the subject does not have a known human immunodeficiency virus (HIV) infection or acute or chronic hepatitis B or C infection. In some embodiments, the individual has a known human immunodeficiency virus (HIV) infection with no detectable viral load or has acute or chronic hepatitis B or C infection with no detectable viral load. In some embodiments, the individual is not immunocompromised. In some embodiments, the individual does not have a history of interstitial lung disease or radiation pneumonitis requiring steroid therapy, or any evidence of clinically active interstitial lung disease or pneumonitis. In some embodiments, the individual does not have uncontrolled diabetes. Exemplary routes of administration, dosages, and frequencies of administration Compositions comprising nanoparticles comprising an mTOR inhibitor and albumin

In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered subcutaneously. In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered intravenously. In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered, for example, via intravenous infusion at a dose between about 1 mg/m ² and about 150 mg/m ² , about 5 mg/m ² and about 75 mg/m ² . In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered, for example, via intravenous infusion, at a dose of about any one of 5, 7.5, 10, 15, 30, 56, 75, or 100 mg/m ^2. In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered to an individual having cancer in one or more 21-day cycles (e.g., three cycles). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered to the subject once during each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered during the 1st, 2nd, or 3rd week of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered on day 1, day 8, or day 15 of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered to the subject twice during each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered between the 1st and 2nd weeks of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered between the 2nd and 3rd weeks of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered between the 1st and 3rd weeks of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered on day 1 and day 8 of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered on day 1 and day 15 of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered on day 8 and day 15 of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered to the subject three times during each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered during week 1, week 2, and week 3 of each 21-day cycle (e.g., three-week cycle). In some embodiments, the mTOR inhibitor nanoparticle composition (e.g., a sirolimus/albumin nanoparticle composition, such as FYARRO™) is administered on day 1, day 8, and day 15 of each 21-day cycle (e.g., a three-week cycle). In some embodiments, the dose of the mTOR inhibitor nanoparticle composition (e.g., a sirolimus/albumin nanoparticle composition, such as FYARRO™) is modified (e.g., if the individual experiences one or more adverse reactions). Details regarding dose modifications of FYARRO™ and the circumstances under which dose modifications are made are described in detail at www.accessdata.fda.gov/drugsatfda_docs/label/2021/213312lbl.pdf. KRAS inhibitors

In some embodiments, the KRAS inhibitor (e.g., KRAS G12C inhibitor) is administered orally. In some embodiments, the KRAS inhibitor (e.g., KRAS G12C inhibitor) is administered in an amount between about 100 mg and about 1200 mg, between about 200 mg and about 1150 mg, between about 300 mg and about 1000 mg, between about 400 mg and about 1000 mg, between about 400 mg and about 960 mg, or between about 400 mg and about 800 mg.

In some embodiments, the KRAS inhibitor is a KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is sotolacib. In some embodiments, sotolacib is administered once daily. In some embodiments, sotolacib is administered (e.g., orally) in an amount between about 100 mg and about 1200 mg, between about 200 mg and about 1150 mg, or between about 300 mg and about 1000 mg. In some embodiments, sotolacib is administered (e.g., orally) in an amount of about 960 mg. In some embodiments, the dosage of sotolacib is modified. Details regarding dose modifications for sotolacilib and circumstances in which dose modifications are made are described in detail at www.accessdata.fda.gov/drugsatfda_docs/label/2021/214665s000lbl.pdf. In some embodiments, the KRAS G12C inhibitor is adagracib. In some embodiments, adagracib is administered twice daily (e.g., bis en die or "BID"). In some embodiments, adagracib is administered in an amount between about 100 mg and about 1200 mg, between about 200 mg and about 1150 mg, between about 300 mg and about 1000 mg, between about 400 mg and about 1000 mg, between about 400 mg and about 960 mg, or between about 400 mg and about 800 mg. In some embodiments, adagracib is administered (e.g., orally) in an amount of about any of 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, 1,100 mg, and 1,200 mg. Exemplary Methods

In some embodiments, a method of treating cancer in an individual (e.g., a human) is provided, comprising administering to the individual (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin ("mTOR inhibitor nanoparticle composition"), wherein the mTOR inhibitor in the nanoparticles is conjugated to albumin (e.g., coated with albumin); and (b) an effective amount of a KRAS G12C inhibitor. In some embodiments, the cancer comprises one or more cells expressing a KRAS G12C mutant protein. Additionally or alternatively, in some embodiments, the cancer comprises one or more cells having at least one mTOR activating aberration. In some embodiments, the cancer is lung cancer (e.g., NSCLC), bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, or a tumor of unknown origin. In some embodiments, the cancer is NSCLC or bladder cancer. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor are administered simultaneously. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor are administered sequentially. In some embodiments, the mTOR inhibitor nanoparticle composition is administered before the KRAS G12C inhibitor. In some embodiments of the treatment method, the KRAS G12C inhibitor is administered before the mTOR inhibitor nanoparticle composition. In some embodiments, the mTOR inhibitor is an mTOR inhibitor described herein. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the mTOR inhibitor is sirolimus (rapamycin) or a derivative or analog thereof. In some embodiments, the mTOR inhibitor nanoparticle composition comprises nanoparticle albumin-bound sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is nanoparticle albumin-bound sirolimus. In some embodiments, the albumin is human albumin, such as human serum albumin. In some embodiments, the KRAS G12C inhibitor is a small molecule inhibitor. In some embodiments, the KRAS G12C inhibitor is sotolacib, as shown below:

In some embodiments, the KRAS G12C inhibitor is adagracib, as shown below:

In some embodiments, the method comprises (such as further comprising) selecting an individual for treatment based on the presence of one or more cancer cells having at least one mTOR activating aberration in a sample (e.g., a tumor sample or a blood sample) from the individual before administering an mTOR inhibitor nanoparticle composition and a KRAS G12C inhibitor. In some embodiments, the method comprises (such as further comprising) selecting an individual for treatment based on the presence of one or more cancer cells expressing a KRAS G12C mutant protein in a sample (e.g., a tumor sample or a blood sample) from the individual before administering an mTOR inhibitor nanoparticle composition and a KRAS G12C inhibitor.

In some embodiments, a method of treating cancer in an individual (e.g., a human) is provided, comprising administering to the individual (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin ("mTOR inhibitor nanoparticle composition"), wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm); and (b) an effective amount of a KRAS G12C inhibitor. In some embodiments, the cancer comprises one or more cells expressing a KRAS G12C mutant protein. Additionally or alternatively, in some embodiments, the cancer comprises one or more cells having at least one mTOR activating aberration. In some embodiments, the cancer is lung cancer (e.g., NSCLC), bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, or a tumor of unknown origin. In some embodiments, the cancer is NSCLC or bladder cancer. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor are administered simultaneously. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor are administered sequentially. In some embodiments, the mTOR inhibitor nanoparticle composition is administered before the KRAS G12C inhibitor. In some embodiments of the treatment method, the KRAS G12C inhibitor is administered before the mTOR inhibitor nanoparticle composition. In some embodiments, the mTOR inhibitor is an mTOR inhibitor described herein. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the mTOR inhibitor is sirolimus (rapamycin) or a derivative or analog thereof. In some embodiments, the mTOR inhibitor nanoparticle composition comprises nanoparticle albumin-bound sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is nanoparticle albumin-bound sirolimus. In some embodiments, the albumin is human albumin, such as human serum albumin. In some embodiments, the KRAS G12C inhibitor is a small molecule inhibitor. In some embodiments, the KRAS G12C inhibitor is sotolacib, the structure of which is shown above. In some embodiments, the KRAS G12C inhibitor is adagracib, the structure of which is shown above. In some embodiments, the method comprises (such as further comprising) selecting an individual for treatment based on the presence of one or more cancer cells having at least one mTOR activating aberration in a sample (e.g., a tumor sample or a blood sample) from the individual before administering an mTOR inhibitor nanoparticle composition and a KRAS G12C inhibitor. In some embodiments, the method comprises (such as further comprising) selecting an individual for treatment based on the presence of one or more cancer cells expressing a KRAS G12C mutant protein in a sample (e.g., a tumor sample or a blood sample) from the individual before administering an mTOR inhibitor nanoparticle composition and a KRAS G12C inhibitor.

In some embodiments, a method of treating cancer in an individual (e.g., a human) is provided, comprising administering to the individual (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin ("mTOR inhibitor nanoparticle composition"), wherein the nanoparticles comprise an mTOR inhibitor conjugated to albumin (e.g., coated with albumin), and wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm); and (b) an effective amount of a KRAS G12C inhibitor. In some embodiments, the cancer comprises one or more cells expressing a KRAS G12C mutant protein. Additionally or alternatively, in some embodiments, the cancer comprises one or more cells having at least one mTOR activating aberration. In some embodiments, the cancer is lung cancer (e.g., NSCLC), bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, or a tumor of unknown origin. In some embodiments, the cancer is NSCLC or bladder cancer. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor are administered simultaneously. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor are administered sequentially. In some embodiments, the mTOR inhibitor nanoparticle composition is administered before the KRAS G12C inhibitor. In some embodiments of the treatment method, the KRAS G12C inhibitor is administered before the mTOR inhibitor nanoparticle composition. In some embodiments, the mTOR inhibitor is an mTOR inhibitor described herein. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the mTOR inhibitor is sirolimus (rapamycin) or a derivative or analog thereof. In some embodiments, the mTOR inhibitor nanoparticle composition comprises nanoparticle albumin-bound sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is nanoparticle albumin-bound sirolimus. In some embodiments, the albumin is human albumin, such as human serum albumin. In some embodiments, the KRAS G12C inhibitor is a small molecule inhibitor. In some embodiments, the KRAS G12C inhibitor is sotolacib, the structure of which is shown above. In some embodiments, the KRAS G12C inhibitor is adagracib, the structure of which is shown above. In some embodiments, the method comprises (such as further comprising) selecting an individual for treatment based on the presence of one or more cancer cells having at least one mTOR activating aberration in a sample (e.g., a tumor sample or a blood sample) from the individual before administering an mTOR inhibitor nanoparticle composition and a KRAS G12C inhibitor. In some embodiments, the method comprises (such as further comprising) selecting an individual for treatment based on the presence of one or more cancer cells expressing a KRAS G12C mutant protein in a sample (e.g., a tumor sample or a blood sample) from the individual before administering an mTOR inhibitor nanoparticle composition and a KRAS G12C inhibitor.

In some embodiments, a method of treating cancer in an individual (e.g., a human) is provided, comprising administering to the individual (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin ("mTOR inhibitor nanoparticle composition"), wherein the weight ratio of albumin to mTOR inhibitor in the mTOR inhibitor nanoparticle composition is about 10:1 (e.g., about 10:1 or about 9:1 or about 8:1); and (b) an effective amount of a KRAS G12C inhibitor. In some embodiments, the cancer comprises one or more cells expressing a KRAS G12C mutant protein. Additionally or alternatively, in some embodiments, the cancer comprises one or more cells having at least one mTOR activating aberration. In some embodiments, the cancer is lung cancer (e.g., NSCLC), bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, or a tumor of unknown origin. In some embodiments, the cancer is NSCLC or bladder cancer. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor are administered simultaneously. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor are administered sequentially. In some embodiments, the mTOR inhibitor nanoparticle composition is administered before the KRAS G12C inhibitor. In some embodiments of the treatment method, the KRAS G12C inhibitor is administered before the mTOR inhibitor nanoparticle composition. In some embodiments, the mTOR inhibitor is an mTOR inhibitor described herein. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the mTOR inhibitor is sirolimus (rapamycin) or a derivative or analog thereof. In some embodiments, the mTOR inhibitor nanoparticle composition comprises nanoparticle albumin-bound sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is nanoparticle albumin-bound sirolimus. In some embodiments, the albumin is human albumin, such as human serum albumin. In some embodiments, the KRAS G12C inhibitor is a small molecule inhibitor. In some embodiments, the KRAS G12C inhibitor is sotolacib, the structure of which is shown above. In some embodiments, the KRAS G12C inhibitor is adagracib, the structure of which is shown above. In some embodiments, the method comprises (such as further comprising) selecting an individual for treatment based on the presence of one or more cancer cells having at least one mTOR activating aberration in a sample (e.g., a tumor sample or a blood sample) from the individual before administering an mTOR inhibitor nanoparticle composition and a KRAS G12C inhibitor. In some embodiments, the method comprises (such as further comprising) selecting an individual for treatment based on the presence of one or more cancer cells expressing a KRAS G12C mutant protein in a sample (e.g., a tumor sample or a blood sample) from the individual before administering an mTOR inhibitor nanoparticle composition and a KRAS G12C inhibitor.

In some embodiments, a method of treating cancer in an individual (e.g., a human) is provided, comprising administering to the individual (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin ("mTOR inhibitor nanoparticle composition"), wherein the nanoparticles comprise an mTOR inhibitor associated with albumin (e.g., coated with albumin), wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm), and wherein the weight ratio of albumin to mTOR inhibitor in the mTOR inhibitor nanoparticle composition is about 10:1 (e.g., about 10:1 or about 9:1 or about 8:1); and (b) an effective amount of a KRAS G12C inhibitor. In some embodiments, the cancer comprises one or more cells expressing a KRAS G12C mutant protein. Additionally or alternatively, in some embodiments, the cancer comprises one or more cells having at least one mTOR activation aberration. In some embodiments, the cancer is lung cancer (e.g., NSCLC), bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, or a tumor of unknown origin. In some embodiments, the cancer is NSCLC or bladder cancer. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor are administered simultaneously. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor are administered sequentially. In some embodiments, the mTOR inhibitor nanoparticle composition is administered before the KRAS G12C inhibitor. In some embodiments of the treatment method, the KRAS G12C inhibitor is administered before the mTOR inhibitor nanoparticle composition. In some embodiments, the mTOR inhibitor is an mTOR inhibitor described herein. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the mTOR inhibitor is sirolimus (rapamycin) or a derivative or analog thereof. In some embodiments, the mTOR inhibitor nanoparticle composition comprises nanoparticle albumin-bound sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is nanoparticle albumin-bound sirolimus. In some embodiments, the albumin is human albumin, such as human serum albumin. In some embodiments, the KRAS G12C inhibitor is a small molecule inhibitor. In some embodiments, the KRAS G12C inhibitor is sotolacib, the structure of which is shown above. In some embodiments, the KRAS G12C inhibitor is adagracib, the structure of which is shown above. In some embodiments, the method comprises (e.g., further comprises) selecting an individual for treatment based on the presence of one or more cancer cells in a sample (e.g., a tumor sample or a blood sample) from the individual prior to administering the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor. In some embodiments, the method comprises (e.g., further comprises) selecting an individual for treatment based on the presence of one or more cancer cells expressing a KRAS G12C mutant protein in a sample (e.g., a tumor sample or a blood sample) from the individual prior to administering the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor.

In some embodiments, a use is provided of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin ("mTOR inhibitor nanoparticle composition") in the manufacture of a medicament for treating cancer in an individual (e.g., a human), wherein the nanoparticles comprise an mTOR inhibitor associated with albumin (e.g., coated with albumin), wherein the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm), and/or wherein the weight ratio of albumin to mTOR inhibitor in the mTOR inhibitor nanoparticle composition is about 10:1 (e.g., about 10:1 or about 9:1 or about 8:1), wherein the medicament is for administration together with a KRAS G12C inhibitor. In some embodiments, a use of a KRAS G12C inhibitor in the manufacture of a medicament for treating cancer in an individual (e.g., a human) is provided, wherein the medicament is for administration with an mTOR inhibitor nanoparticle composition, wherein the nanoparticle comprises an mTOR inhibitor associated with albumin (e.g., coated with albumin), wherein the nanoparticle has an average particle size of no more than about 150 nm (e.g., no more than about 120 nm), and/or wherein the weight ratio of albumin to mTOR inhibitor in the mTOR inhibitor nanoparticle composition is about 10:1 (e.g., about 10:1 or about 9:1 or about 8:1). In some embodiments, the cancer comprises one or more cells expressing a KRAS G12C mutant protein. Additionally or alternatively, in some embodiments, the cancer comprises one or more cells having at least one mTOR activating aberration. In some embodiments, the cancer is lung cancer (e.g., NSCLC), bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, or a tumor of unknown origin. In some embodiments, the cancer is NSCLC or bladder cancer. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor are administered simultaneously. In some embodiments, the mTOR inhibitor nanoparticle composition and the KRAS G12C inhibitor are administered sequentially. In some embodiments, the mTOR inhibitor nanoparticle composition is administered before the KRAS G12C inhibitor. In some embodiments of the treatment method, the KRAS G12C inhibitor is administered before the mTOR inhibitor nanoparticle composition. In some embodiments, the mTOR inhibitor is an mTOR inhibitor described herein. In some embodiments, the mTOR inhibitor is a limus drug. In some embodiments, the mTOR inhibitor is sirolimus (rapamycin) or a derivative or analog thereof. In some embodiments, the mTOR inhibitor nanoparticle composition comprises nanoparticle albumin-bound sirolimus. In some embodiments, the mTOR inhibitor nanoparticle composition is nanoparticle albumin-bound sirolimus. In some embodiments, the albumin is human albumin, such as human serum albumin. In some embodiments, the KRAS G12C inhibitor is a small molecule inhibitor. In some embodiments, the KRAS G12C inhibitor is sotolacib, the structure of which is shown above. In some embodiments, the KRAS G12C inhibitor is adagracib, the structure of which is shown above. In some embodiments, the method comprises (such as further comprising) selecting an individual for treatment based on the presence of one or more cancer cells having at least one mTOR activating aberration in a sample from the individual (e.g., a tumor sample or a blood sample) before administering an mTOR inhibitor nanoparticle composition and a KRAS G12C inhibitor. In some embodiments, the method comprises (such as further comprising) selecting an individual for treatment based on the presence of one or more cancer cells expressing KRAS G12C mutant protein in a sample from the individual (e.g., a tumor sample or a blood sample) before administering an mTOR inhibitor nanoparticle composition and a KRAS G12C inhibitor. Products and Kits

An article of manufacture comprising a material suitable for treating cancer in an individual is provided. In certain embodiments, the article of manufacture or kit comprises a container containing a composition comprising nanoparticles comprising an mTOR inhibitor and albumin ("mTOR inhibitor nanoparticle composition"). In certain embodiments, the mTOR inhibitor is an mTOR inhibitor described herein. In certain embodiments, the mTOR inhibitor is a limus drug. In certain embodiments, the mTOR inhibitor is sirolimus (rapamycin) or a derivative or analog thereof. In certain embodiments, the mTOR inhibitor nanoparticle composition comprises nanoparticle albumin-bound sirolimus. In certain embodiments, the mTOR inhibitor nanoparticle composition is nanoparticle albumin-bound sirolimus. In some embodiments, the nanoparticles comprise an mTOR inhibitor conjugated to albumin (e.g., coated with albumin). In some embodiments, the nanoparticles have an average particle size of no more than about 150 nm (e.g., no more than about 120 nm). In some embodiments, the weight ratio of albumin to mTOR inhibitor in the mTOR inhibitor nanoparticle composition is about 10:1 or less (e.g., about 10:1 or about 9:1 or about 8:1). In some embodiments, the albumin is human albumin, such as human serum albumin. In some embodiments, the kit includes one or more positive controls, such as cells having at least one mTOR activation aberration (for additional details on mTOR activation aberrations, see elsewhere herein). In some embodiments, the kit includes negative controls, such as cells that do not have any mTOR activation aberrations. In some embodiments, the kit is used to treat a cancer comprising a cell that expresses a KRAS mutant protein (e.g., a KRAS G12C mutant protein, a KRAS G12A mutant protein, a KRAS G12D mutant protein, a KRAS G12F mutant protein, a KRAS G12L mutant protein, a KRAS G12R mutant protein, a KRAS G12S mutant protein, a KRAS G12V mutant protein, a KRAS G13A mutant protein, a KRAS G13C mutant protein, a KRAS G13D mutant protein, a KRAS G13P mutant protein, a KRAS G13R mutant protein, a KRAS G13S mutant protein, a KRAS G13V mutant protein, a KRAS Q61E mutant protein, a KRAS Q61H mutant protein, a KRAS Q61K mutant protein, a KRAS Q61L mutant protein, a KRAS In some embodiments, the kit is used to treat cancer comprising one or more cells having at least one mTOR activating aberration. In some embodiments, the cancer is a solid tumor, lung cancer, bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, uterine cancer, endometrial cancer, cervical cancer, testicular cancer, bile duct cancer, myelodysplastic cancer, or a tumor of unknown origin. In some embodiments, the cancer is a solid tumor, lung cancer, or bladder cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC).

In certain embodiments, the article or kit comprises a container and a label or package insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, test tubes, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition (e.g., an mTOR inhibitor nanoparticle composition described herein, such as nanoparticle albumin-bound sirolimus), alone or in combination with another composition that is effective in treating cancer (e.g., delaying the progression of cancer). In some embodiments, the cancer comprises a cell line that expresses a KRAS mutant protein (e.g., a KRAS G12C mutant protein, a KRAS G12A mutant protein, a KRAS G12D mutant protein, a KRAS G12F mutant protein, a KRAS G12L mutant protein, a KRAS G12R mutant protein, a KRAS G12S mutant protein, a KRAS G12V mutant protein, a KRAS G13A mutant protein, a KRAS G13C mutant protein, a KRAS G13D mutant protein, a KRAS G13P mutant protein, a KRAS G13R mutant protein, a KRAS G13S mutant protein, a KRAS G13V mutant protein, a KRAS Q61E mutant protein, a KRAS Q61H mutant protein, a KRAS Q61K mutant protein, a KRAS Q61L mutant protein, a KRAS Q61P mutant protein, a KRAS In some embodiments, the cancer comprises one or more cells having at least one mTOR activation aberration. In some embodiments, the cancer comprises one or more cells having at least one mTOR activation aberration. In some embodiments, the cancer is a solid tumor, lung cancer, bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, uterine cancer, endometrial cancer, cervical cancer, testicular cancer, bile duct cancer, myelodysplastic cancer, or a tumor of unknown origin. In some embodiments, the cancer is a solid tumor, lung cancer, or bladder cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). The container may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one agent in the composition is an mTOR inhibitor nanoparticle composition described herein, such as nanoparticle albumin-bound sirolimus. In some embodiments, the label or package insert indicates that the composition is used in combination with a KRAS inhibitor (e.g., a KRAS G12C inhibitor, a KRAS G12A inhibitor, a KRAS G12D inhibitor, a KRAS G12F inhibitor, a KRAS G12L inhibitor, a KRAS G12R inhibitor, a KRAS G12S inhibitor, a KRAS G12V inhibitor, a KRAS G13A inhibitor, a KRAS G13C inhibitor, a KRAS G13D inhibitor, a KRAS G13P inhibitor, a KRAS G13R inhibitor, a KRAS G13S inhibitor, a KRAS G13V inhibitor, a KRAS Q61E inhibitor, a KRAS KRAS A146E inhibitor, KRAS A146G inhibitor, KRAS A146P inhibitor, KRAS A146S inhibitor, KRAS A146T inhibitor or KRAS A146V inhibitor). In some embodiments, the KRAS inhibitor is a KRAS G12C inhibitor, such as but not limited to sotolacib, also known as AMG 510 (Amgen/Beiji Shenzhou); MRTX849, also known as adagracib (Mirati/Zai Lab), JAB-21822 (Jacob Pharmaceuticals), GDC-6036 (Genentech), JDQ443 (Novartis), D-1553 (Invitrogen and Merck), GH35 (Qinhao Pharmaceuticals), GFH925 (Jinfang Pharmaceuticals), BPI-421286 (Beida Pharmaceuticals), LY3537982, RMC-6291 (Revolution Medicine), RMC-8839 (Revolution Medicine), HBI-2438 (Shanghai Asia Bioscience International Co., Ltd.) and JNJ-74699157 (Johnson & Johnson). In some embodiments, the KRAS inhibitor is a KRAS G12D inhibitor, such as but not limited to MRTX1133 (Mirati Therapeutics) or RMC-6236 (Revolution Medicines). In some embodiments, the KRAS inhibitor is a KRAS G12V inhibitor, such as but not limited to JAB-23000.

In some embodiments, the package insert provided with the product or kit contains information regarding the indications, usage, dosage, administration, contraindications and/or warnings regarding one or more compositions provided with the product or kit.

In addition, the product or kit may include (a) a first container containing a composition, wherein the composition includes an mTOR inhibitor nanoparticle composition described herein, such as nanoparticle albumin-bound sirolimus; and (b) a second container containing a composition, wherein the composition includes a KRAS inhibitor (e.g., a polypeptide, antibody, fusion polypeptide, antisense oligonucleotide, or small molecule drug capable of inhibiting the activity of a KRAS mutant protein described herein). In some embodiments, the second container contains a small molecule KRAS inhibitor (e.g., a KRAS inhibitor described herein). In addition, the product may further include an additional container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The kits may optionally provide additional components such as buffers and descriptive information. Thus, the present application also provides articles of manufacture that include vials (e.g., sealed vials), bottles, jars, flexible packaging, and the like.

Those skilled in the art will recognize that within the scope and spirit of the present invention, several embodiments are possible. The present invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the present invention, but of course should not be construed as limiting its scope in any way.

All publications and patent applications cited in this specification are incorporated herein by reference, just as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Examples

The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Every effort has been made to ensure accuracy of numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weights are weight average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Example 1: Antitumor activity of a combination of nanoparticle albumin-bound sirolimus and a KRAS G12C inhibitor in mice bearing human non-small cell lung cancer (NSCLC) tumor xenografts

In this example, the anti-tumor efficacy of each of ( i ) nanoparticle albumin-bound sirolimus, ( ii ) everolimus (i.e., a sirolimus derivative), ( iii ) sotolacib (i.e., a small molecule KRAS G12C inhibitor), ( iv ) nanoparticle albumin-bound sirolimus + sotolacib, and ( v ) everolimus + sotolacib was evaluated in mice bearing NCI-H2030 tumor xenografts. NCI-H2030 is a human non-small cell lung cancer (NSCLC) adenocarcinoma cell line with KRAS ^G12C and STK11 ^{E317 *} mutations. (NCI-H2030 mutation distribution: KRAS ^G12C , STK11 ^{E317 *} , TP53 ^G262V .) The KRAS G12C mutation, which is common in NSCLC, leads to constitutive activation of the RAS/MAPK signaling pathway, which promotes tumor growth. STK11 is a negative regulator of mTOR signaling. The mTOR pathway is often activated in patients with KRAS mutations and contributes to adaptive resistance to KRAS inhibitors.

Female athymic mice were implanted bilaterally with human NCI-H2030 NSCLC cells. When tumors reached approximately 100-150 ^mm3 , animals were randomly divided into five groups (n = 3/group) and administered with saline (control), single agent drugs, or drug combinations as shown in Table A below, according to the route, dose, and frequency of administration shown in Table A1 below. Additional information regarding the treatment regimens in Table A1 is provided in Table A2 . Table A1 - Treatment Groups for the NCI-H2030 Tumor Xenograft Model Group Mouse number Tumor One or more medications One or more doses One or more routes of administration Frequency 1 3 NCI-H2030 Salt water (control) Not applicable IV* 10 ml 2 times a week 2 3 NCI-H2030 Nanoparticle albumin-bound sirolimus 7.5 mg/kg IV Twice a week 3 3 NCI-H2030 Everolimus 3 mg/kg PO** qdx5 per week† 4 3 NCI-H2030 Sotolacib 30 mg/kg PO qdx5 per week 5 3 NCI-H2030 Nanoparticle albumin-bound sirolimus + sotolacib 7.5 mg/kg + 30 mg/kg IV + PO 2 times a week + qdx5 per week 6 3 NCI-H2030 Everolimus + Sotolacizumab 3 mg/kg + 30 mg/kg PO + PO qdx5 per week + qdx5 per week * IV = intravenous ** PO = oral (i.e., administered by mouth) † Once daily, 5 times per week Table A2 - Additional information for treatment regimens in Tables A1, D, and F Material Dose / ^frequencya Weekly dose (mg /kg ) Clinical weekly medication % Way Salt water 10 mL /kg twice a week 0 NA IV Nanoparticle albumin-bound sirolimus 7.5 mg /kg twice a week 15 45 IV Everolimus 3 mg /kg , 5 days / week 15 115 PO Sotolacizumab ^* 30 mg /kg , 5 days / week 150 11 PO Adagracib ^* 30 mg /kg , 5 days / week 150 9 PO ^a Dosages used in previous nonclinical studies. Dosing was once daily for 6 weeks; dosing schedule was consistent across models. IV = intravenous Nab = nanoparticle albumin-bound PO = oral * Martin et al., American Association for Cancer Research 2022 Annual Meeting, Announcement 2670

As shown in Figure 1A , the NABI-sirolimus + sotolacib combination demonstrated significantly greater tumor growth inhibition or "TGI" (109%) compared to single-agent NABI-sirolimus (68%, P = 0.0102, ANOVA), everolimus (22%, P = 0.0011), or sotolacib (20%, P = 0.0002), and the combination of everolimus and sotolacib (53%, P = 0.0008) in the NCI-H2030 model. Table B shows that the combination of NABP-sirolimus and sotolacib was the only treatment that showed a statistically significant difference in tumor growth inhibition (TGI) compared to saline and all other treatment groups (two-way ANOVA). No other treatment was found to show a statistically significant TGI compared to saline or other treatment groups. Table B: Two-way ANOVA for TGI NABP-sirolimus + sotolacib vs. other treatment groups Comparison of TGI ( treatment group vs. treatment group ) p -value Nanoparticle albumin-bound sirolimus + sotolacib versus saline 0.0003 Nanoparticle albumin-bound sirolimus + sotolacib vs. nanoparticle albumin-bound sirolimus 0.0102 Nanoparticle albumin-bound sirolimus + sotolacib versus everolimus 0.0011 Nanoparticle albumin-bound sirolimus + sotolacib vs sotolacib 0.0002 Nanoparticle albumin-bound sirolimus + sotolacib versus everolimus + sotolacib 0.0008

Figure 1B provides a waterfall plot showing tumor volume regression in NCI-H2030 xenograft mice treated with saline, NAb-sirolimus, everolimus, sotolacizumab, NAb-sirolimus + sotolacizumab, and everolimus + sotolacizumab. The greater TGI observed in mice treated with NAb-sirolimus + sotolacizumab was associated with a significantly higher tumor response rate (tumor regression of more than -30% change in tumor volume) in the NAb-sirolimus + sotolacizumab treatment group compared to all other treatment groups (P = 0.0059, chi-square). Table C shows the response rates (i.e., tumor regression > 30%) in NCI-H2030 xenograft mice in treatment groups 1 to 6 from Table A. Tumor regression (e.g., tumor volume change > -30%) was observed only in mice treated with nanoparticle albumin-bound sirolimus + sotolacib. Combination treatment with everolimus + sotolacib did not improve tumor response rates compared to single-agent everolimus or single-agent sotolacib. Table C. Response rates in NCI-H2030 xenograft mice (P = 0.0602) Group One or more medications Response rate % ( tumor regression > - 30 % ) 1 Salt water (control) 0 2 Nanoparticle albumin-bound sirolimus 0 3 Everolimus 0 4 Sotolacib 0 5 Nanoparticle albumin-bound sirolimus + sotolacib 50% 6 Everolimus + Sotolacib 0

When tumor growth curves were compared, statistical significance was observed for NAb-sirolimus plus sotolacib versus single-agent NAb-sirolimus ( P = 0.0001) or sotolacib ( P < 0.0001) and the combination of everolimus plus sotolacib ( P = 0.0036).

All treatments were well tolerated with no signs of toxicity, and body weight changes were similar to those of saline controls (see Figure 1C ).

Next, a similar set of experiments was performed in mice carrying human NCI-H2122 tumor xenografts. NCI-H2122 is a human NSCLC squamous cell carcinoma cell line with KRAS ^G12C and STK11 null (loss-of-function) mutations. (NCI-H2122 mutation profile: NCI-H2122: KRAS ^G12C , STK11 ^null , TP53 ^C176F .) Briefly, female athymic mice were implanted with NCI-H2122 cells on both flanks. When tumor volume reached approximately 100-150 ^mm3 , animals were randomly divided into nine groups (n = 5/group) and administered with saline (control), single agent drugs, or drug combinations as shown in Table D below, according to the route of administration, dose, and frequency of administration shown in Table D below. Adalicib is a small molecule KRAS G12C inhibitor. Additional information about the treatment regimens in Table D is provided in Table A2 . Table D: Treatment Groups for the NCI-H2122 Tumor Xenograft Model Group Mouse number Tumor One or more medications One or more doses One or more routes of administration Frequency 1 5 NCI-H2122 Salt water (control) Not applicable IV ^* Twice a week 2 5 NCI-H2122 Nanoparticle albumin-bound sirolimus 7.5 mg/kg IV Twice a week 3 5 NCI-H2122 Everolimus 3 mg/kg PO ^** qdx5 per week† 4 5 NCI-H2122 Sotolacib 30 mg/kg PO qdx5 per week 5 5 NCI-H2122 Adagracib 30 mg/kg PO qdx5 per week 6 5 NCI-H2122 Nanoparticle albumin-bound sirolimus + sotolacib 7.5 mg/kg + 30 mg/kg IV + PO 2 times a week + qdx5 per week 7 5 NCI-H2122 Everolimus + Sotolacizumab 3 mg/kg + 30 mg/kg PO + PO qdx5 per week + qdx5 per week 8 5 NCI-H2122 Nanoparticle albumin-bound sirolimus + adagracib 7.5 mg/kg + 30 mg/kg IV + PO 2 times a week + qdx5 per week 9 5 NCI-H2122 Everolimus + Adalicibub 3 mg/kg + 30 mg/kg PO + PO qdx5 per week + qdx5 per week * IV = intravenous ** PO = oral (i.e., administered by mouth) † Once daily, 5 times per week

As shown in Figure 2A , combination treatment with an mTOR inhibitor and a KRAS G12C inhibitor was significantly better in inhibiting NCI-H2122 tumor growth (TGI = 103%) compared to treatment with single-agent nanoparticle albumin-bound sirolimus (TGI = 80%, P = 0.0001, ANOVA) or single-agent sotolacib (79%, P < 0.0001). The combination of nanoparticle albumin-bound sirolimus + adagracib showed a significantly greater TGI (99%) compared to single-agent nanoparticle albumin-bound sirolimus (80%, P = 0.0002) or single-agent adagracib (58%, P < 0.0001). Single-agent sotolacib was significantly more active in the NCI-H2122 model compared with adagraciib (P = 0.0143). Unexpectedly, there was no significant difference in TGI between NAb-sirolimus + sotolacib vs NAb-sirolimus vs adagraciib. In addition, NAb-sirolimus + sotolacib and NAb-sirolimus + adagraciib were statistically superior in inhibiting tumor growth compared with everolimus + sotolacib and everolimus + adagraciib, respectively: NAb-sirolimus + sotolacib vs. everolimus + sotolacib: P = 0.0036 (ANOVA); NAb-sirolimus + adagraciib vs. everolimus + adagraciib: P = 0.0013; NAb-sirolimus + sotolacib vs. NAb-sirolimus + adagraciib: P = not significant (ns).

FIG. 2B provides a waterfall graph showing tumor volume regression in NCI-H2122 xenograft mice treated with saline, NAb-sirolimus, everolimus, sotolacizumab, adagracib, NAb-sirolimus + sotolacizumab, everolimus + sotolacizumab, NAb-sirolimus + adagracib, and everolimus + adagracib. Improved TGI in mice treated with NAb-sirolimus plus sotolacib or NAb-sirolimus plus adagraciib was associated with significantly higher tumor response rates (e.g., tumor regression with a change of more than -30% in tumor volume) compared with saline and all single-agent treatments (P < 0.0001, chi-square, and P = 0.0347, respectively). Tumor regression (e.g., tumor volume change greater than -30%) was 80% in the NAb-sirolimus plus sotolacib group and 20% in the everolimus plus sotolacib group (P = 0.0230, Fisher's exact test). The combination of everolimus plus sotolacib ( Fig. 2B ) did not significantly increase the rate of tumor regression compared with single-agent treatment with either agent. Tumor regression was numerically higher in the NAb-sirolimus plus adagracib group compared with the everolimus plus adagracib group (40% vs. 0%, respectively, P = 0.0867). Table E shows the response rates (i.e., tumor regression >30%) in NCI-H2122 xenograft mice in treatment groups 1 to 9 from Table D. The response rates in mice treated with single-agent nanoparticle albumin-bound sirolimus or single-agent everolimus were 10% and 20%, respectively, indicating that each single agent exhibited little anti-tumor activity. However, it was unexpectedly found that the response rates of mice treated with a combination of nanoparticle albumin-bound sirolimus and sotolacizumab or adagracib (i.e., 80% and 40%, respectively) were significantly higher than the response rates of mice treated with a combination of everolimus and sotolacizumab or adagracib (i.e., 20% and 0%, respectively). Table E. Response rate of NCI-H2122 xenograft mice (P = not significant) Group One or more medications Response rate % ( tumor regression > - 30 % ) 1 Salt water (control) 0 2 Nanoparticle albumin-bound sirolimus 20% 3 Everolimus 10% 4 Sotolacib 0% 5 Adagracib 0% 6 Nanoparticle albumin-bound sirolimus + sotolacib 80% 7 Everolimus + Sotolacib 20% 8 Nanoparticle albumin-bound sirolimus + adagracib 40% 9 Everolimus + adaglacirib 0%

All treatment groups showed improved survival (i.e., number of days) compared to saline controls. The median survival time of mice given saline was 16 days. The median survival time of mice in the other treatment groups was not reached (p < 0.0001, log-rank test).

All treatments were tolerable with no signs of toxicity, and body weight changes were similar to saline controls (see Figure 2C ). Example 2: Antitumor activity of the combination of nanoparticle albumin-bound sirolimus and KRAS G12C inhibitor in mice bearing human bladder cancer tumor xenografts

In this example, the anti-tumor efficacy of each of ( i ) nanoparticle albumin-bound sirolimus, ( ii ) sotolacib, ( iii ) adagracib, ( iv ) nanoparticle albumin-bound sirolimus + sotolacib, and ( v ) nanoparticle albumin-bound sirolimus + adagracib was evaluated in mice bearing UMUC3 tumor xenografts. UMUC3 is a human metastatic cell carcinoma (bladder cancer) cell line with KRAS ^G12C and PTEN null mutations. PTEN is a negative regulator or mTOR activity (UMUC3 mutation distribution: KRAS ^G12C , PTEN ^null , TP53 ^F113C , ATM ^Q2800fs , CDKN2A ^null , UGT2B17 ^null ).

Briefly, female athymic mice were implanted with NCI-H2122 cells in their right flank. When tumor volume reached approximately 100-150 ^mm3 , animals were randomly divided into nine groups (n = 6/group) and administered with saline (control), single agent drugs, or drug combinations as shown in Table F below according to the route of administration, dose, and frequency of administration shown in Table F below. Additional information about the treatment regimens in Table F is provided in Table A2 . Table F: Treatment Groups of UMUC3 Tumor Xenograft Model Group Mouse number Tumor One or more medications One or more doses One or more routes of administration Frequency 1 6 UMUC3 Salt water (control) Not applicable IV ^* Twice a week 2 6 UMUC3 Nanoparticle albumin-bound sirolimus 7.5 mg/kg IV Twice a week 3 6 UMUC3 Sotolacib 3 mg/kg PO ^** qdx5 per week† 4 6 UMUC3 Adagracib 30 mg/kg PO qdx5 per week 5 6 UMUC3 Nanoparticle albumin-bound sirolimus + sotolacib 7.5 mg/kg + 30 mg/kg IV + PO 2 times a week + qdx5 per week 6 6 UMUC3 Nanoparticle albumin-bound sirolimus + adagracib 7.5 mg/kg + 30 mg/kg IV + PO 2 times a week + qdx5 per week * IV = intravenous ** PO = oral (i.e., administered by mouth) † Once daily, 5 times per week

None of the mice (0/6) survived to the end of the study (i.e., day 42) in any of the saline control, nanoparticle albumin-bound sirolimus, and sotolacizumab groups. None of the mice (0/6) were tumor-free in any of these groups. Among mice treated with adagracib, 1/6 survived to day 42, and 3/6 were tumor-free during the study (median tumor-free time: 24 days; range: 13-29 days). Among mice treated with nanoparticle albumin-bound sirolimus + sotolacizumab, 3/6 survived to day 42, and 6/6 mice were tumor-free during the study (median tumor-free time: 28 days; range: 25-34 days). Of the mice treated with NABI-sirolimus + adagracib, 3/6 survived to day 42, and 5/6 were tumor-free during the study (median tumor-free time: 31 days; range: 19-34 days). See Figure 3A . Treatment of mice with NABI-sirolimus + sotogracib or NABI-sirolimus + adagracib resulted in almost complete elimination of UMUC3 tumors (complete response, 6/6 mice treated with NABI-sirolimus + sotogracib and 5/6 mice treated with NABI-sirolimus + adagracib, respectively). The combination of NABI-sirolimus and either KRAS inhibitor, sotolacizumab or adagraciib, showed significantly greater tumor growth inhibition compared to single agent NABI-sirolimus, sotolacizumab or adagraciib. See Figure 3A . There was no significant difference in tumor growth inhibition between the combination of NABI-sirolimus and sotolacizumab or adagraciib (see Figure 3A ).

Mice treated with NAb-sirolimus + sotolacib exhibited significantly greater TGI (105%) compared to mice treated with single-agent NAb-sirolimus (79%, P < 0.0001, ANOVA) or single-agent sotolacib (100%, P < 0.0001). Mice treated with NAb-sirolimus + adagraciib exhibited significantly greater TGI (105%) compared to mice treated with single-agent NAb-sirolimus (79%, P < 0.0001) or single-agent adagraciib (103%, P = 0.0374). Compared to the results from the NCI-H2122 model (see Figure 2A ), single-agent adagracib was significantly more active than sotogracib in the UMUC3 model (P = 0.0035). Unexpectedly, no significant difference in TGI was observed in mice treated with nanoparticle albumin-bound sirolimus + sotogracib vs. mice treated with nanoparticle albumin-bound sirolimus + adagracib. No significant difference in TGI was observed in mice treated with nanoparticle albumin-bound sirolimus + sotogracib vs. mice treated with nanoparticle albumin-bound sirolimus + adagracib.

The greater TGI observed in mice treated with NABI-sirolimus + sotolacib or NABI-sirolimus + adagracib was associated with significantly higher tumor response rates (e.g., tumor regression of more than -30% tumor volume change) compared to saline and single agents (P < 0.0001, chi-square and P = 0.0005, respectively). See Figure 3B . Table G shows the response rates (i.e., tumor regression > 30%) in UMUC3 xenograft mice in treatment groups 1 to 6 from Table F. Table F: Response rate of UMUC3 xenograft mice (P < 0.0001 for nanoparticle albumin-bound sirolimus + sotolacib; P = 0.0005 for nanoparticle albumin-bound sirolimus + adagraciib) Group One or more medications Response rate % ( tumor regression > - 30 % ) 1 Salt water (control) 0 2 Nanoparticle albumin-bound sirolimus 0% 3 Sotolacib 0% 4 Adagracib 50% 5 Nanoparticle albumin-bound sirolimus + sotolacib 100% 6 Nanoparticle albumin-bound sirolimus + adagracib 100%

Mice treated with NABP-sirolimus plus sotolacib showed improved survival compared to mice treated with sotolacib alone (p = 0.0183 (log rank)). Additionally, mice treated with NABP-sirolimus plus adagracib showed improved survival compared to mice treated with adagracib alone (p = 0.0708 (log rank)).

All treatments were well tolerated with no signs of toxicity, and weight changes were similar to saline controls (see Figure 3C ). Example 3: Comparison of antitumor activity of single-agent sotolacib versus single-agent adagracib and nanoparticle albumin-bound sirolimus + sotolacib versus nanoparticle albumin-bound sirolimus + adagracib combination therapy

Figure 4 shows the anti-tumor activity of single-agent sotolacib and single-agent adagraciib in mice bearing NCI-H2122 NSCLC tumors (left side, data from Figure 2A ) and single-agent sotolacib and single-agent adagraciib in mice bearing UMUC3 tumors (right side, data from Figure 3A ). Sotolacib was more effective than adagraciib in the NCI-H2122 model (P = 0.0143), while adagraciib was more effective in the UMUC3 model (P = 0.0035).

Without theoretical constraints, the relative efficacy of sotolacizumab and adagracib may be tumor-specific. However, in all tumor models tested in Examples 1 and 2, the combination of nanoparticle albumin-bound sirolimus + sotolacizumab and nanoparticle albumin-bound sirolimus + adagracib exhibited significantly improved anti-tumor activity compared to single-agent nanoparticle albumin-bound sirolimus, single-agent sotolacizumab, and single-agent adagracib. See Figure 1A , Figure 2A , and Figure 3A . Regardless of the relative antitumor activity of each single agent in NSCLC and bladder cancer xenograft models, there was no difference in the antitumor efficacy of nanoparticle albumin-bound sirolimus + sotolacib and nanoparticle albumin-bound sirolimus + adagraciib in either model. See, e.g., Figure 2A and Figure 3A .

Treatment with NABI-sirolimus + sotorasib and NABI-sirolimus + adagraciib significantly increased the rate of significant tumor regression in all 3 models tested, as analyzed by significant tumor response or rate of significant tumor regression (e.g., greater than 30% reduction in tumor size), not only compared to treatment with single-agent NABI-sirolimus, single-agent everolimus, single-agent sotorasib, or single-agent adagraciib, but also compared to treatment with everolimus + sotorasib or everolimus + adagraciib. Combination treatment with everolimus + sotolacib or everolimus + adagracib inhibited (e.g., slowed) tumor growth, but everolimus + KRAS G12C inhibitor combination treatment did not result in any increase in the rate of tumor regression in any of the models tested.

In the H2030 model, the antitumor activity of single - agent nanoparticle albumin-bound sirolimus was numerically superior to that of single-agent everolimus (see Figures 1A and 1B ). In contrast, there was no significant difference in the antitumor activity of single-agent nanoparticle albumin-bound sirolimus and single-agent everolimus in the H2122 model (see Figures 2A and 2B ). Unexpectedly, the response rate (e.g., tumor regression of >-30% of tumor volume) was significantly higher in mice treated with a combination of nanoparticle albumin-bound sirolimus and sotolacizumab or adagracib than in mice treated with a combination of everolimus and sotolacizumab or adagracib.

Overall, nanoparticle albumin-bound sirolimus + KRAS inhibitor induced partial responses (PR) and complete responses (CR), while everolimus + KRAS inhibitor induced some mice with stable disease (SD) and mice with disease progression.

As shown in Examples 1 to 3, nanoparticle albumin-bound sirolimus exhibited synergistic antitumor activity when combined with sotolacizumab or adagracib compared to either agent alone, as well as significantly enhanced tumor growth inhibition and significant tumor regression. This study demonstrated consistent tumor growth inhibition and significantly higher tumor drug levels were observed with nanoparticle albumin-bound sirolimus compared to everolimus. See, e.g., Figure 6. All treatments were tolerable, with no overt signs of toxicity and produced similar weight change patterns compared to saline controls in each study. The results suggest that nanoparticle albumin-bound sirolimus should be a better mTOR inhibitor in clinical practice for combination therapy with KRAS inhibitors (e.g., adagracib or sotolaccib). Example 4: Phase 1/2 trial of a combination of a KRAS G12C inhibitor and an mTOR nanoparticle composition in patients with advanced solid tumors with KRAS G12C mutations and non-small cell lung cancer (NSCLC) with KRAS G12C mutations Overview

This Phase 1/2 study evaluates the safety and clinical activity of a KRAS G12C inhibitor (e.g., adagraciib or sotograciib) in combination with ABI-009 (i.e., an exemplary sirolimus/albumin nanoparticle combination, also known as nanoparticle albumin-bound sirolimus and FYARRO™) in a patient population with advanced solid tumors/NSCLC with KRAS G12C mutations who have received prior therapy in the advanced or metastatic setting. Target Population

The target population is patients with advanced, unresectable or metastatic solid tumors or NSCLC with KRAS G12C mutation. Number of patients in the trial

The trial will enroll approximately 50-90 patients: • Phase 1 : Approximately 15-25 patients with solid tumors • Phase 2 cohorts: o Cohort A : Approximately 20-40 NSCLC patients not previously exposed to KRAS G12C inhibitors o Cohort B : Approximately 20-40 NSCLC patients previously exposed to KRAS G12C inhibitors (a) Objectives and Indicators Phase 1 - Objectives

The primary objectives of the Phase 1 portion of the trial include, but are not limited to, ( i ) characterizing the safety and tolerability of the combination of a KRAS G12C inhibitor and the mTOR nanoparticle combination ABI-009 (also known as FYARRO® and nanoparticle albumin-bound sirolimus) in patients with advanced solid tumors harboring a KRAS G12C mutation, and ( ii ) establishing the maximum tolerated dose (MTD) of the combination using one or more dosing regimens and/or identifying a recommended Phase 2 combination dose (RP2D) and regimen for a KRAS G12C inhibitor and ABI-009.

Secondary objectives of the Phase 1 portion of the trial include ( i ) evaluating the pharmacokinetics (PK) of the KRAS G12C inhibitor and ABI-009 when administered in combination and ( ii ) evaluating the clinical activity (e.g., clinical efficacy) of the combination of the KRAS G12C inhibitor and ABI-009 in patients with malignant solid tumors harboring KRAS G12C mutations.

The exploratory objectives of the Phase 1 portion of the trial include, but are not limited to, ( i ) exploring potential pharmacodynamic markers of signal transduction inhibition in tumor tissue; ( ii ) evaluating the utility of detecting KRAS G12C mutations in plasma to identify the study population; and ( iii ) exploring the correlation between tumor biomarkers, genetic alterations, and efficacy. Phase 1 - Indications

The primary endpoints of the Phase 1 portion of the trial include, but are not limited to, ( i ) safety, characterized by the type, incidence, severity, timing, relationship of severity to study treatment of adverse events (AEs) and laboratory abnormalities, and the number of patients who modified or discontinued study treatment due to adverse events (AEs) from the first dose of study treatment to 28 days after the last dose of study treatment; and ( ii ) the maximum tolerated dose (MTD) and/or recommended Phase 2 dose (RP2D) of the KRAS G12C inhibitor and ABI-009 administered in combination. Safety parameters are used to assess the number of patients with dose-limiting toxicity (DLT) to determine the MTD/RP2D.

Secondary endpoints of the Phase 1 portion of the trial include, but are not limited to, ( i ) plasma pharmacokinetic parameters of KRAS G12C inhibitor and ABI-009, ( ii ) objective response rate (ORR) defined by RECIST 1.1 (see Eisenhauer et al. (2009) Eur J. Cancer 45: 228-247), ( iii ) duration of response (DOR), ( iv ) progression-free survival (PFS), ( v ) PFS at 6 months and 12 months , ( vi ) 1-year survival rate, and ( vii ) overall survival (OS).

Overall survival (OS) is usually measured as the time from the start of autonomous treatment to the date of death from any cause. Progression-free survival (PFS) is usually measured as the time from the start of autonomous treatment to the date of progressive disease (PD) or death from any cause, whichever comes first. Duration of response (DOR) is usually measured as the time from the start of autonomous treatment to the first documented objective tumor response (complete response ("CR") or partial response ("PR")) to the first documented PD or death from any cause, whichever comes first. Objective response rate (ORR) is usually measured as the proportion of individuals (e.g., patients, individuals) who experience a confirmed complete response (CR) or partial response (PR) based on RECIST v1.1 criteria during the time period from the first treatment dose until the last treatment dose. Exploratory endpoints for the Phase 1 portion of the trial include, but are not limited to, ( i ) levels of KRAS G12C protein modification, ( ii ) the kinetics of genetic alterations in tumor tissue and circulating tumor DNA (ctDNA) and the concordance between KRAS G12C mutations identified in tumor tissue versus ctDNA, and ( iii ) mutations in RAS and other tumor genes that may be involved in sensitivity and resistance to the combination of KRAS G12C inhibitors and ABI-009, including TSC1 and TSC2 mutations. Phase 2 - Objectives

The primary objectives of the Phase 2 portion of the trial include, but are not limited to, evaluating the clinical efficacy of a KRAS G12C inhibitor in combination with ABI-009 in patients with NSCLC harboring a KRAS G12C mutation.

Secondary objectives of the Phase 2 portion of the trial include, but are not limited to, ( i ) evaluating the pharmacokinetics of KRAS G12C inhibitors and ABI-009 when administered in combination, ( ii ) characterizing the safety and tolerability of the combination of KRAS G12C inhibitors and ABI-009 in patients with NSCLC harboring KRAS G12C mutations, and ( iii ) evaluating the clinical activity of the combination of KRAS G12C inhibitors and ABI-009 in patients with solid malignant tumors harboring KRAS G12C mutations.

The exploratory objectives of the Phase 2 portion of the trial include, but are not limited to, ( i ) exploring potential pharmacodynamic markers of signal transduction inhibition in tumor tissue; ( ii ) evaluating the utility of detecting KRAS G12C mutations in plasma to identify the study population; and ( iii ) exploring the correlation between tumor biomarkers, genetic alterations, and efficacy. Phase 2 - Indications

The primary endpoint of the Phase 2 portion of the trial includes, but is not limited to, clinical efficacy based on ORR as defined by RECIST 1.1 (see Eisenhauer et al. (2009) Eur J. Cancer 45: 228-247).

Secondary endpoints for the Phase 2 portion of the trial include, but are not limited to, ( i ) plasma pharmacokinetic concentrations of KRAS G12C inhibitor and ABI-009, ( ii ) safety, characterized by the type, incidence, severity, timing, relationship of severity to study treatment of AEs and laboratory abnormalities, and the number of patients who modified or discontinued study treatment due to adverse events from the first dose of study treatment to 28 days after the last dose of study treatment, ( iii ) duration of response (DOR), ( iv ) progression-free survival (PFS), ( v ) PFS at 6 and 12 months, ( vi ) 1-year survival rate, and ( vii ) overall survival (OS). Overall survival (OS) is usually measured as the time from the start of autonomous treatment to the date of death from any cause. Progression-free survival (PFS) is usually measured as the time from the start of autonomous treatment to the date of progressive disease (PD) or death from any cause, whichever comes first. Duration of response (DOR) is usually measured as the time from the start of autonomous treatment to the first documented objective tumor response (complete response ("CR") or partial response ("PR")) to the first documented PD or death from any cause, whichever comes first. Objective response rate (ORR) is usually measured as the proportion of individuals (e.g., patients, individuals) who experience a confirmed complete response (CR) or partial response (PR) based on RECIST v1.1 criteria during the time period from the first treatment dose until the last treatment dose.

Exploratory endpoints for the Phase 2 portion of the trial include, but are not limited to, ( i ) levels of KRAS G12C protein modification, ( ii ) the kinetics of gene alterations in tumor tissue and ctDNA and the concordance between KRAS G12C mutations identified in tumor tissue versus ctDNA, and ( iii ) mutations in RAS and other tumor genes that may be involved in sensitivity and resistance to the combination of KRAS G12C inhibitors and ABI-009, including TSC1 and TSC2 mutations. (b) Study Design

This example describes a Phase 1/2 evaluation of the safety, pharmacokinetics, and clinical activity of a combination of a KRAS G12C inhibitor and ABI-009 in patients with advanced malignant solid tumors with KRAS G12C mutations and NSCLC with KRAS G12C mutations.

Study treatment (i.e., KRAS G12C inhibitor in combination with ABI-009) is administered in 3-week cycles (or according to an alternative schedule). KRAS G12C inhibitor is administered orally continuously daily (or according to an alternative schedule). KRAS G12C inhibitor is administered orally twice daily (i.e., bis en die or "BID") at a dose between 200 mg and 800 mg or once daily (i.e., "qd") at a dose between 100 mg and 2000 mg. ABI-009 is administered by intravenous (IV) infusion on Days 1 and 8 every 21 days (i.e., twice every three weeks). Alternatively, ABI-009 is administered once a week or once every three weeks. ABI-009 is administered at doses between 1 mg/m ² and 75 mg/m ^2. Dosing may be escalated or decreased depending on toxicity experienced.

Patients received study treatment at the discretion of the investigator until disease progression, unacceptable adverse events, patient refusal, or death. Phase 1 - Study Design

The study started with a pharmacokinetic (PK) lead-in of the first dose level of KRAS G12C inhibitor and ABI-009 to evaluate the PK of the two agents when given in combination. Approximately 24 patients participated. The dosing schedule for the PK lead-in is provided in Table G1 and the dosing schedule for Cycle 1 and all subsequent cycles is provided in Table G2 below, and the overall study plan is provided in Figure 5. Table G1 PK introduction Study Days 1 2 3 4 5 6 7 8 9 10 11 12 13 14 ABI - 009 Medication Administration X KRAS G12C inhibitor dosing X X X X X X X Table G2 First and subsequent cycles Study Days 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 twenty one ABI - 009 Medication Administration X X KRAS G12C inhibitor dosing X X X X X X X X X X X X X X X X X X X X X

For any specific regimen of KRAS G12C inhibitor and ABI-009 administered, the MTD of the combination regimen is the dose associated with a target toxicity rate of 0.30 during the first treatment cycle, with an acceptable toxicity probability interval of (0.25, 0.35). Phase 2 - Study Design

After determination of the MTD and/or potential feasible RP2D regimen, additional patients were enrolled in the Phase 2 cohorts for a total of approximately 55 NSCLC patients (approximately 30 patients in Cohort A and approximately 25 patients in Cohort B) to further assess safety/tolerability and clinical activity.

If warranted, patients were added to the additional dose confirmation/expansion cohort. Objective response rate (ORR) according to RECIST 1.1 (see Eisenhauer et al. (2009) Eur J Cancer. 45(2): 228-47) was the clinical activity marker for hypothesis testing. Patients experiencing clinical benefit could continue study treatment after progressive disease as defined by RECIST 1.1 at the discretion of the investigator. Patients who discontinued treatment were followed up for subsequent anticancer therapy and survival. (c) Patient selection and inclusion criteria Inclusion criteria

Inclusion criteria for this Phase 1/2 clinical study are: • Histologically confirmed: o Phase 1 : Histologically confirmed malignant solid tumor with KRAS G12C mutation in tumor tissue or plasma ctDNA. o Phase 2 : Histologically confirmed NSCLC with KRAS G12C mutation in tumor tissue or plasma ctDNA. • Unresectable or metastatic disease. • Not a candidate for definitive therapy (e.g., curative-intent therapy is not available); in addition: • Phase 2 patients (Cohorts A and B) must have received prior therapy with platinum compounds and checkpoint inhibitors (with any curative intent). • Patients in Cohort A must have never received a prior KRAS G12C inhibitor. • Patients in Cohort B must have received a prior KRAS G12C inhibitor. • Presence of measurable disease per RECIST 1.1. • Age ≥ 18 years. • Life expectancy of at least 3 months. • Recent prior systemic therapy (e.g., chemotherapy, immunotherapy, or investigational agents) and radiation therapy interrupted for at least 2 weeks prior to the first dose of study treatment. • Recovery from adverse reactions to prior therapy to ≤ Grade 1 (excluding alopecia, peripheral neuropathy, and parameters substituted by other eligibility criteria, such as hematologic parameters) at the time of enrollment. • Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 (see, e.g., www.ecog-acrin.org/resources/ecog-performance-status). o Adequate organ function as determined by laboratory values during the screening period: o Absolute neutrophil count ≥ 1,500/mm ³ (≥ 1.5 × 10 ⁹ /L); o Platelet count ≥ 100,000/mm ³ (≥ 100 × 10 ⁹ /L); o Hemoglobin ≥ 9 g/dL in the absence of transfusions for at least 2 weeks; o Total bilirubin ≤ 1.5 × upper limit of normal (ULN) (≤ 3 × ULN if associated with liver metastases or Gilbert's disease); o Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 3.0 × ULN (≤ 5 × ULN if associated with liver metastases); o Creatinine clearance ≥ 50 mL/min or glomerular filtration rate ≥ 50 mL/min/1.73 m ² calculated using validated prediction equations (e.g., Cockcroft-Gault, Modification of Diet in Nephropathy (MDRD) or 24-hour urine output CrCl). • Women of childbearing potential (WOCBP) or men with WOCBP partners agreed to use contraception while enrolled in this study and for a period of 6 months after discontinuation of study treatment. Exclusion Criteria • Active brain metastases or carcinomatous meningitis. Patients with brain metastases were eligible if: o Brain metastases were adequately treated and o Patients were neurologically stable for at least 2 weeks prior to enrollment and o Steroid dose ≤ 10 mg pramirama (or equivalent) daily. • History of significant hemoptysis or hemorrhage within 4 weeks of the first dose of study treatment. • Major surgery within 4 weeks of the first dose of study treatment. • History of intestinal disease, inflammatory bowel disease, major gastric surgery, or other gastrointestinal pathology that could alter absorption of study treatment or lead to inability to swallow oral medications (e.g., uncontrolled nausea, vomiting, malabsorption syndrome). • Any of the following cardiac abnormalities in the last 6 months prior to participation: o Unstable angina or myocardial infarction; o Congestive heart failure ≥ New York Heart Association stage 3; o Prolonged corrected QT (QTc) > 480 ms on ECG during the screening period or medical or family history of congenital long QT syndrome; o Symptomatic or uncontrolled atrial flutter or other clinically significant arrhythmia. • History of stroke or transient ischemic attack in the past 6 months. • Continuing need for medications with a known risk of torsades de pointes or narrowly treated CYP3A substrates; strong inhibitors or inducers of CYP3A and/or P-gp; strong inhibitors of breast cancer resistance protein (BCRP); and proton pump inhibitors, who were unable to switch to alternative therapy prior to study entry. At least 5 half-lives must have elapsed since discontinuation of these concomitant medications prior to initiation of study treatment. • Known or suspected presence of another malignancy that could be mistaken for the malignancy being studied during disease evaluation. • Known human immunodeficiency virus (HIV) infection or acute or chronic hepatitis B or C infection. Patients who are allowed to be treated for hepatitis C with undetectable viral load and patients who are treated for HIV with undetectable viral load for at least 1 month. • Immunocompromised patients (currently receiving immunosuppression for solid organ or bone marrow transplant patients, patients with congenital immunodeficiency, or patients who are deemed by the investigator to be at risk for opportunistic infections by immunosuppressive medications). • Received a live vaccine within 14 days of the first dose of study treatment. • History of interstitial lung disease or radiation pneumonitis requiring steroid therapy, or any evidence of clinically active interstitial lung disease or pneumonitis. • Uncontrolled diabetes within 4 weeks of study treatment. • Known hypersensitivity to KRAS G12C inhibitors or ABI-009 or any formulation. • Pregnancy (WOCBP must have documented a negative serum or urine pregnancy test during the screening period prior to the start of study drug). • Breastfeeding or planning to breastfeed during the study or for 6 months after study treatment. • Any serious illness, uncontrolled intermittent illness, psychiatric disorder, active or uncontrolled infection, or other medical history, including laboratory results, that the investigator believes may interfere with the patient’s participation in the study or the interpretation of the results. Dietary Restrictions

Patients should avoid the following substances due to their potential pharmacokinetic interactions with KRAS G12C inhibitors and/or ABI-009: • Grapefruit juice; • Curcumin/turmeric; and • Herba Lycopodii and other herbal preparations. (d) Patient treatment

The combination therapy (i.e., KRAS G12C inhibitor and ABI-009) was administered in 21-day cycles. For the PK run-in cycle only, the KRAS G12C inhibitor was administered orally starting on Day 8, and ABI-009 was administered intravenously on Day 1 for 30 minutes. For all other cycles, the KRAS G12C inhibitor was administered orally starting on Day 1, and ABI-009 was administered intravenously on Days 1 and 8 for 30 minutes. The KRAS G12C inhibitor was administered before ABI-009. The KRAS G12C inhibitor was administered twice daily, 12 hours apart whenever possible. ABI-009 was dosed based on body surface area. Dosing was based on the patient's height at study entry and weight at study entry, and no dose adjustment was made unless the patient's weight changed by more than 5% from baseline. Phase 1 Segment

The study began by evaluating the combination of a KRAS G12C inhibitor administered at a dose between 200 mg and 800 mg BID (i.e., twice daily) and ABI-009 at a dose between 1 mg/m ² and 75 mg/m ^2. The combination regimen is administered in 21-day cycles. The regimen of the KRAS G12C inhibitor and/or ABI-009 is adjusted based on observed toxicity, toxicity analysis and/or PK profile. Other doses of the KRAS G12C inhibitor and/or ABI-009 may be explored depending on emerging data. The doses of both the KRAS G12C inhibitor and ABI-009 may be reduced as needed to manage adverse events (AEs) and seek an RP2D regimen. Regimens that can be used in Phase 2

The combination of a KRAS G12C inhibitor and ABI-009 is considered Phase 2-ready, characterized by adequate tolerability, with at least 80% of the intended dose intensity expected to be delivered during at least 2 treatment cycles and must be at or below the maximum tolerated dose (defined as the dose associated with a target toxicity rate of 0.30 during the first treatment cycle, with an acceptable toxicity probability interval of (0.25, 0.35)). Phase 2 Dose Confirmation/Expansion Segment

The Phase 2 portion of the study evaluated the clinical efficacy of the combination of a KRAS G12C inhibitor and ABI-009 in a population of NSCLC patients with KRAS G12C mutations and specified tumor histology, treatment history, and baseline characteristics (see Patient Selection and Eligibility Criteria). Patients received treatment with a KRAS G12C inhibitor and ABI-009 using the dose levels and schedules determined in the dose escalation portion of the study. (e) Efficacy Indicator Definition and Analysis Objective Response Rate (ORR)

Objective disease response was classified according to RECIST 1.1 criteria (see Eisenhauer et al. (2009) Eur J Cancer. 45(2): 228-47). Objective response rate was determined as the percentage of patients with a documented confirmed complete response (CR) or partial response (PR). Duration of response (DOR)

Duration of response was defined as the time from the date of the first documented objective tumor response (CR or PR) to the first documented objective progressive disease (PD) or death from any cause in the absence of documented PD. The Kaplan Meier method was used for the subgroup of patients with objective responses to obtain an estimate of the median DOR. Progression-free survival (PFS)

Progression-free survival was determined as the time from the date of first study treatment to the first PD or death from any cause if no PD was documented. The Kaplan-Meier method was used to obtain an estimate of the median PFS time. Overall Survival (OS)

Time to death was defined as the time from the first date of study treatment to death from any cause. Kaplan-Meier was used to estimate median OS and 1-year survival; 95% confidence intervals for 1-year survival were reported. Subgroup Analysis

Baseline characteristics from phase 2 assessed in subgroup analyses included sex, age, smoking status, and the source of tumor DNA used to detect the KRAS G12C mutation (e.g., tumor tissue or ctDNA).

PK evaluable population pharmacokinetic parameters were determined using standard non-compartmental methods. PK parameters include, but are not limited to, the following: • Cmax (ng/mL) : Maximum plasma concentration of KRAS G12C inhibitor and/or ABI-009 observed during the dosing interval • Cmin (ng/mL) : Minimum observed concentration of KRAS G12C inhibitor and/or ABI-009 during the dosing interval • tmax (hr) : Time to maximum plasma concentration of KRAS G12C inhibitor and/or ABI-009 observed during the dosing interval • t½ (hr) : Terminal elimination half-life of KRAS G12C inhibitor and/or ABI-009 • AUClast (ng*h/mL) : KRAS The area under the plasma concentration-time curve of KRAS G12C inhibitor and/or ABI-009 from time zero to the last measurable time point, calculated by log-linear trapezoidal summation. • AUC∞ (ng*h/mL) : The area under the plasma concentration-time curve of KRAS G12C inhibitor and/or ABI-009 from time zero to infinity, calculated by log-linear trapezoidal summation and extrapolated to infinity by adding the last observed quantifiable plasma concentration divided by the elimination rate constant λz (AUC0-∞ is not reported if %AUCext ＞ 20%). • CL/F (L/hr) : Apparent clearance of KRAS G12C inhibitor after oral administration. • Vz/F (L) : KRAS Apparent volume of distribution of G12C inhibitors after oral administration • CL (L/hr) : Apparent systemic clearance of KRAS G12C inhibitors and/or ABI-009 in plasma.

The present invention has been described with respect to specific embodiments discovered or proposed by the inventors to include the best mode for practicing the invention. Those skilled in the art will appreciate that, in accordance with the present invention, many modifications and changes may be made in the specific embodiments illustrated without departing from the intended scope of the invention. All such modifications are intended to be included within the scope of the accompanying claims. Example 5

In an analysis of colorectal cancer patients treated with nanoparticle albumin-bound rapamycin, it was found that patients with KRAS wild-type responded better than those with KRAS mutations (overall response rate 44% vs. 20%). The results suggest that mutations in the KRAS pathway lead to resistance to nanoparticle albumin-bound sirolimus in cancer patients with activated mTOR pathways. Therefore, a combination strategy of KRAS/NRAS inhibitors may be required for patients with such mutations. Example 6

method:

Mice bearing NCI-H2122 tumors were treated for 6 weeks or until tumor size exceeded 2000 mm ³ . At the end of the study, tumors were harvested and analyzed for biomarkers by Western blotting.

result:

After long-term treatment, nanoparticle albumin-bound sirolimus alone or in combination with sotolacib or adagracib showed stronger inhibition of mTORC1 target phosphorylated S6 compared to the corresponding everolimus alone or in combination groups. See Figure 7A to Figure 7B. In addition, nanoparticle albumin-bound sirolimus alone also showed stronger inhibition of mTORC1 target phosphorylated 4EBP1 compared to everolimus alone. The results confirmed that nanoparticle albumin-bound sirolimus alone or in combination with KRAS inhibitors strongly inhibited mTORC1 activity.

FIG. 1A provides the results of experiments performed to evaluate the antitumor activity of ( i ) nanoparticle albumin-bound sirolimus, ( ii ) everolimus, ( iii ) sotolacib, ( iv ) nanoparticle albumin-bound sirolimus + sotolacib, and ( v ) everolimus + sotolacib in mice bearing NCI-H2030 human non-small cell lung cancer xenografts.

FIG. 1B provides a waterfall graph showing tumor volume regression in NCI-H2030 xenograft mice treated with saline, NABI-sirolimus, everolimus, sotolaciib, NABI-sirolimus + sotolaciib, and everolimus + sotolaciib.

FIG. 1C shows the % change in body weight of NCI-H2030 xenograft mice treated with saline, NABI-sirolimus, everolimus, sotolacib, NABI-sirolimus + sotolacib, and everolimus + sotolacib.

FIG. 2A provides the results of experiments performed to evaluate the anti-tumor activity of ( i ) nanoparticle albumin-bound sirolimus, ( ii ) everolimus, ( iii ) sotolacizumab, ( iv ) adagracib, ( v ) nanoparticle albumin-bound sirolimus + sotolacizumab, ( vi ) everolimus + sotolacizumab, ( viii ) nanoparticle albumin-bound sirolimus + adagracib, and ( viii ) everolimus + adagracib in mice bearing NCI-H2122 human non-small cell lung cancer xenografts.

FIG. 2B provides a waterfall graph showing tumor volume regression in NCI-H2122 xenograft mice treated with saline, NAb-sirolimus, everolimus, sotolacizumab, adagracib, NAb-sirolimus + sotolacizumab, everolimus + sotolacizumab, NAb-sirolimus + adagracib, and everolimus + adagracib.

Figure 2C shows the % weight change of NCI-H2122 xenograft mice treated with saline, NAb-sirolimus, everolimus, sotolacizumab, adagracib, NAb-sirolimus + sotolacizumab, everolimus + sotolacizumab, NAb-sirolimus + adagracib, and everolimus + adagracib.

FIG3A provides the results of experiments performed to evaluate the anti-tumor activity of ( i ) nanoparticle albumin-bound sirolimus, ( ii ) sotolacizumab, ( iii ) adagracib, ( iv ) nanoparticle albumin-bound sirolimus + sotolacizumab, and ( v ) nanoparticle albumin-bound sirolimus + adagracib in mice bearing UMUC3 human bladder cancer xenografts.

FIG3B provides waterfall graphs showing tumor volume regression in UMUC3 xenograft mice treated with saline, NABI-sirolimus, sotolacizumab, adagracib, NABI-sirolimus + sotolacizumab, and NABI-sirolimus + adagracib.

FIG. 3C shows the % weight change of UMUC3 xenograft mice treated with saline, NABI-sirolimus, sotolacizumab, adagracib, NABI-sirolimus + sotolacizumab, and NABI-sirolimus + adagracib.

FIG4 shows the anti-tumor activity of single -agent sotolacizumab and single-agent adagraciib in mice bearing NCI-H2122 NSCLC tumors (left side, data obtained from FIG2A ) and the anti-tumor activity of single-agent sotolacizumab and single-agent adagraciib in mice bearing UMUC3 tumors (right side, data obtained from FIG3A ).

Figure 5 shows the study design for the Phase 1 and Phase 2 portions of the clinical trial described in Example 4.

6A - 6B show a comparison of (A) tumor and (B) blood concentrations of nanoparticle albumin-bound sirolimus and everolimus in the NSCLC (adenocarcinoma) NCI-H2122 model described in Example 1.

FIG. 7A - B show the results of Western blot analysis of phospho-S6 and phosphor-4EBP1 in tumor cells after treatment with nanoparticle albumin-bound sirolimus, everolimus, or a combination of nanoparticle albumin-bound sirolimus/everolimus and one of sotolacizumab or adagraciba.

Claims (54)

A method for treating cancer in an individual, comprising administering to the individual: (a) an effective amount of a composition comprising nanoparticles comprising an mTOR inhibitor and albumin, and (b) an effective amount of a KRAS inhibitor. The method of claim 1, wherein the mTOR inhibitor is limus drug. The method of claim 2, wherein the limus drug is sirolimus. The method of any one of claims 1 to 3, wherein the average diameter of the nanoparticles in the composition is no more than about 150 nm. The method of claim 4, wherein the average diameter of the nanoparticles in the composition does not exceed about 120 nm. The method of any one of claims 1 to 5, wherein the weight ratio of the albumin to the mTOR inhibitor in the nanoparticle composition does not exceed about 10:1. The method of any one of claims 1 to 6, wherein the nanoparticles comprise the mTOR inhibitor conjugated to the albumin. The method of claim 7, wherein the nanoparticles comprise the mTOR inhibitor coated with the albumin. The method of any one of claims 1 to 8, wherein the mTOR inhibitor nanoparticle composition is administered intravenously or subcutaneously. The method of claim 9, wherein the mTOR inhibitor nanoparticle composition is administered intravenously. The method of any one of claims 1 to 10, wherein the KRAS inhibitor is an antibody, peptide, protein, antisense oligonucleotide or small molecule that inhibits the activity of a KRAS mutant protein. The method of claim 11, wherein the KRAS inhibitor is a small molecule. The method of claim 12, wherein the KRAS inhibitor is a small molecule KRAS G12C inhibitor selected from the group consisting of: sotolacib, adagracib, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, RMC-8839, HBI-2438 and JNJ-74699157. The method of claim 13, wherein the small molecule KRAS G12C inhibitor is sotolacib or adagraciib. The method of claim 14, wherein the sotolacib or adagracib is administered orally. The method of any one of claims 1 to 15, wherein the cancer comprises one or more cancer cells expressing a KRAS G12C mutant protein. The method of claim 12, wherein the KRAS inhibitor is a small molecule KRAS G12D inhibitor selected from the group consisting of MRTX1133 and RMC-6236. The method of any one of claims 1 to 11 and 17, wherein the cancer comprises one or more cancer cells expressing a KRAS G12D mutant protein. The method of claim 12, wherein the KRAS inhibitor is a small molecule KRAS G12V inhibitor, and wherein the small molecule KRAS G12V inhibitor is JAB-23000. The method of any one of claims 1 to 11 and 19, wherein the cancer comprises one or more cancer cells expressing a KRAS G12V mutant protein. The method of any one of claims 1 to 20, comprising one or more cancer cells expressing a KRAS mutant protein and/or having at least one mTOR activation aberration. The method of any one of claims 1 to 21, wherein the cancer comprising one or more cancer cells expressing KRAS mutant protein and/or having at least one mTOR activation aberration is a solid tumor, lung cancer, bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, uterine cancer, endometrial cancer, cervical cancer, testicular cancer, bile duct cancer, myelodysplastic cancer, or a tumor of unknown origin. The method of claim 22, wherein the cancer is a solid tumor, lung cancer, bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, or a tumor of unknown origin. The method of claim 23, wherein the cancer is a solid tumor, lung cancer, or bladder cancer. The method of claim 23, wherein the solid tumor is an advanced, unresectable and/or metastatic solid tumor. The method of claim 24, wherein the lung cancer is non-small cell lung cancer (NSCLC). The method of claim 26, wherein the NSCLC is advanced, unresectable and/or metastatic NSCLC. The method of any one of claims 1 to 27, wherein the mTOR inhibitor nanoparticle composition and the KRAS inhibitor are administered simultaneously. The method of any one of claims 1 to 27, wherein the mTOR inhibitor nanoparticle composition and the KRAS inhibitor are administered concurrently. The method of any one of claims 1 to 27, wherein the mTOR inhibitor nanoparticle composition and the KRAS inhibitor are administered sequentially. The method of claim 30, wherein the mTOR inhibitor nanoparticle composition is administered once a week, once every three weeks, or twice every three weeks. The method of claim 31, wherein the KRAS inhibitor is administered daily or twice daily. The method of any one of claims 1 to 32, wherein the individual is a human. The method of any one of claims 1 to 33, wherein the method comprises selecting the subject for treatment based on the presence of one or more cancer cells having at least one mTOR activating aberration prior to administering the mTOR inhibitor nanoparticle composition and the KRAS inhibitor. The method of claim 34, wherein the mTOR activation aberration comprises a mutation in an mTOR-related gene. The method of claim 34 or 35, wherein the mTOR activation aberration is in at least one mTOR-related gene selected from the group consisting of: AKT1, FLT-3, MTOR, PIK3CA, PIK3CG, TSC1, TSC2, RHEB, STK11, NF1, NF2, TP53, FGFR4, BAP1, KRAS, NRAS, NRF2, KEAP1 and PTEN. The method of claim 36, wherein the mTOR activation aberration is in TSC1 and/or TSC2. The method of any one of claims 1 to 37, wherein the method comprises selecting the individual for treatment based on the presence of one or more cancer cells expressing a KRAS mutant protein. The method of claim 38, wherein the KRAS mutant protein is a KRAS G12C mutant protein, a KRAS G12D mutant protein, or a KRAS G12V mutant protein. A kit for treating cancer in an individual, comprising: (a) a composition comprising nanoparticles comprising an mTOR inhibitor and albumin, and (b) instructions for administering an effective amount of the mTOR inhibitor nanoparticle composition and an effective amount of a KRAS inhibitor to an individual having cancer, wherein the cancer comprises one or more cancer cells expressing a KRAS mutant protein and/or having at least one mTOR activating aberration. The kit of claim 40, wherein the mTOR inhibitor is the drug limus. The kit of claim 40 or 41, wherein the limus drug is sirolimus. The kit of any one of claims 40 to 42, wherein the average diameter of the nanoparticles in the composition does not exceed about 150 nm. The kit of claim 43, wherein the average diameter of the nanoparticles in the composition does not exceed about 120 nm. The kit of any one of claims 40 to 44, wherein the weight ratio of the albumin to the mTOR inhibitor in the nanoparticle composition does not exceed about 10:1. The kit of any one of claims 40 to 45, wherein the nanoparticles comprise the mTOR inhibitor conjugated to the albumin. The kit of claim 46, wherein the nanoparticles comprise the mTOR inhibitor coated with the albumin. The kit of any one of claims 40 to 47, wherein the KRAS inhibitor is an antibody, peptide, protein, antisense oligonucleotide or small molecule that inhibits the activity of the KRAS mutant protein. The kit of claim 48, wherein the KRAS inhibitor is a small molecule. The kit of claim 49, wherein the KRAS inhibitor is a small molecule KRAS G12C inhibitor selected from the group consisting of: sotolacib, adagracib, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, RMC-8839, HBI-2438 and JNJ-74699157. The kit of claim 50, wherein the KRAS inhibitor is a small molecule KRAS G12D inhibitor selected from the group consisting of MRTX1133 and RMC-6236. The kit of claim 51, wherein the KRAS inhibitor is a small molecule KRAS G12V inhibitor, and wherein the small molecule KRAS G12V inhibitor is JAB-23000. The kit of any one of claims 40 to 52, wherein the cancer is a solid tumor, lung cancer, bladder cancer, coccyx cancer, colorectal cancer, small intestine cancer, pancreatic cancer, uterine cancer, endometrial cancer, cervical cancer, testicular cancer, bile duct cancer, myelodysplastic cancer, or a tumor of unknown origin. A set as in any one of claims 40 to 53, wherein the individual is a human.